Amino acid and peptide conjugates and uses thereof

ABSTRACT

The present invention relates to peptides, and amino acid and peptide conjugates, methods for making amino acid and peptide conjugates, conjugates produced by the methods, pharmaceutical compositions comprising the peptides and conjugates, methods of eliciting immune responses in a subject and methods of vaccinating a subject, uses of the peptides and conjugates for the same, and uses of the peptides and conjugates in the manufacture of medicaments for the same.

This application is a continuation application of U.S. patentapplication Ser. No. 15/535,956, filed Jun. 14, 2017, which is a § 371application of PCT/IB2015/059901, filed Dec. 22, 2015, which in turnclaims priority to U.S. Provisional Application No. 62/096,106, filedDec. 23, 2014. The entire disclosure of each of the foregoingapplications is incorporated by reference herein.

Incorporated herein by reference in its entirety is the Sequence Listingbeing concurrently submitted via EFS-Web as a text file namedSeqList.txt, created Jan. 16, 2019, and having a size of 60,450 bytes.

TECHNICAL FIELD

The present invention relates to amino acid and peptide conjugates,methods for making amino acid and peptide conjugates, conjugatesproduced by the methods, pharmaceutical compositions comprising thepeptides and conjugates, methods of eliciting immune responses in asubject and methods of vaccinating a subject, uses of the peptides andconjugates for the same, and uses of the peptides and conjugates in themanufacture of medicaments for the same.

BACKGROUND ART

Synthetic peptide vaccines generally comprise a synthetic copy of animmunogenic part of protein antigens. This approach to vaccinedevelopment has a number of advantages, including ease of synthesis,avoidance of potentially toxic biological by-products andstraightforward characterisation.

A key issue in the development of peptide vaccines is the lack ofimmunogenicity displayed by peptides as sole vaccine components. It isusually necessary to include in the vaccine an adjuvant, designed toactivate components of the innate immune system (e.g. Freund'sadjuvant).

An alternative strategy in peptide vaccine design is to createself-adjuvanting vaccines in which the peptide epitope of interest iscovalently linked to an appropriate adjuvant.

Such self-adjuvanting vaccines may have enhanced antigen uptake,presentation and dendritic cell maturation compared to simpleco-formulation of the antigen with an external adjuvant.

Several self-adjuvanting vaccines have been developed, but preparationof the vaccines can be complicated.

There is an ongoing need for new self-adjuvanting vaccines and newmethods of making self-adjuvanting vaccines. In particular, there is aneed for self-adjuvanting vaccines directed to treating Epstein BarrVirus (EBV) associated diseases, such as Hodgkin's Disease (HD) orNasopharangeal Carcinoma (NPC), including, for example, self-adjuvatingvaccines comprising one or more epitopes from EBV Latent MembraneProtein 2 (LMP2).

It is an object of the present invention to go some way towards meetingthese needs; and/or to at least provide the public with a useful choice.

Other objects of the invention may become apparent from the followingdescription which is given by way of example only.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or are common general knowledge in the fieldrelevant to the present invention as it existed before the prioritydate.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for making apeptide conjugate, the method comprising

-   -   providing a lipididated amino acid or peptide, and    -   coupling the lipidated amino acid or peptide to one or more        amino acids or peptides to provide a peptide conjugate, and        wherein the peptide conjugate comprises one or more EBV LMP2        epitopes.

In one aspect, the present invention provides a method for making apeptide conjugate, the method comprising reacting

-   -   a lipid-containing conjugation partner, and    -   an amino acid-comprising conjugation partner,    -   under conditions effective to conjugate the lipid-containing        conjugation partner to the amino acid-comprising conjugation        partner by the hydrothiolation of a carbon-carbon double bond        with a thiol,    -   the method further comprising coupling the amino acid of the        amino acid conjugate to an amino acid or a peptide to provide a        peptide conjugate, and wherein the peptide conjugate comprises        one or more EBV LMP2 epitopes.

In another aspect, the present invention provides a method for making apeptide conjugate, the method comprising reacting

-   -   a lipid-containing conjugation partner, and    -   a peptide-comprising conjugation partner, wherein the        peptide-comprising partner comprises one or more EBV LMP2        epitopes,    -   under conditions effective to conjugate the lipid-containing        conjugation partner to the peptide-comprising conjugation        partner by the hydrothiolation of a carbon-carbon double bond        with a thiol.

In another aspect, the present invention provides an amino acidconjugate or peptide conjugate as herein described. In variousembodiments, the amino acid conjugate or peptide conjugate comprises oneor more EBV LMP2 epitopes. In one example, the amino acid conjugate orpeptide conjugate is an amino acid conjugate or peptide conjugate madeby a method of the present invention.

In one embodiment, the present invention provides a peptide conjugatecomprising one or more EBV LMP2 epitopes. In various embodiments, theone or more EBV LMP2 epitopes are MHCI epitopes. In various embodiments,the peptide conjugate comprises one or more EBV LMP2 epitopes selectedfrom the group consisting of any one of SEQ ID NOs 76-101. In variousembodiments, the peptide conjugate comprises a peptide comprising orconsisting of 12 or more contiguous amino acids from the amino acidsequence of any one of SEQ ID NOs 1-75. In various embodiments, the thepeptide conjugate comprises a peptide comprising or consisting of 15 ormore contiguous amino acids from the amino acid sequence of any one ofSEQ ID NOs 1-75, or comprising or consisting of 20 or more contiguousamino acids from the amino acid sequence of any one of SEQ ID NOs 1-75.

In another aspect, the present invention provides an isolated, purified,or recombinant peptide comprising or consisting of 12 or more contiguousamino acids from the amino acid sequence of any one of SEQ ID NOs 1-75.In various embodiments, the isolated, purified, or recombinant peptidecomprises or consists of 15 or more contiguous amino acids from theamino acid sequence of any one of SEQ ID NOs 1-75, or comprises orconsists of 20 or more contiguous amino acids from the amino acidsequence of any one of SEQ ID NOs 1-75.

In one embodiment, the peptide comprises, consists of, or consistsessentially of an amino acid sequence selected from the group consistingof any one of SEQ ID NOs 1-75.

Any of the embodiments described herein relate to any of the aspectsherein.

In various embodiments, the lipidated amino acid or peptide comprisesone or more of the following: Pam1Cys, Pam2Cys, and Pam3Cys. Forexample, the lipidated amino acid or peptide comprises one or more ofthe following: Pam1CSK₄, Pam2 CSK₄, and Pam3 CSK₄. In variousembodiments, the lipidated amino acid or peptide is acetylated oramidated, for example, the lipidated amino acid or peptide comprisesacetylated Pam1Cys, acetylated Pam2Cys, or acetylated Pam3Cys, oramidated Pam1Cys, amidated Pam2Cys, or amidated Pam3Cys.

In one embodiment, the amino acid-comprising conjuation partner is apeptide-containing conjugation partner, and the lipid-containingconjugation partner is coupled to the peptide of the peptide-containingconjugation partner.

In some embodiments, the lipid-containing conjugation partner isconjugated to the or an amino acid of the amino acid-containingconjugation partner or the peptide of the peptide-containing conjugationpartner.

In certain embodiments, the lipid-containing conjugation partner isconjugated to the or an amino acid of the amino acid-containingconjugation partner.

Accordingly, in another aspect, the present invention provides a methodfor making a peptide conjugate, the method comprising reacting

-   -   a lipid-containing conjugation partner, and    -   a peptide-containing conjugation partner, wherein the        peptide-comprising partner    -   comprises one or more EBV LMP2 epitopes,    -   under conditions effective to conjugate the lipid-containing        conjugation partner to the peptide of the peptide-containing        conjugation partner by the hydrothiolation of a carbon-carbon        double bond with a thiol.

In one embodiment, the conjugate is a lipopeptide, such that the methodis for making a lipopeptide.

In one embodiment, the lipid-containing conjugation partner comprisesthe carbon-carbon double bond, and the peptide of the peptide-containingconjugation partner comprises the thiol.

In one embodiment, the amino acid-comprising conjugation partnercomprises one or more EBV LMP2 epitopes. In one embodiment, thepeptide-containing conjugation partner comprises one or more EBV LMP2epitopes. In one embodiment, the amino acid-comprising conjugationpartner comprises two or more EBV LMP2 epitopes. In one embodiment, thepeptide-containing conjugation partner comprises two or more EBV LMP2epitopes. In one embodiment, the peptide conjugate comprises two or moreEBV LMP2 epitopes. In one embodiment, the epitope is a peptide epitope.In one embodiment, the amino acid-comprising conjugation partnerconsists of a peptide. In one embodiment, the amino acid-comprisingconjugation partner consists of a peptide comprising a peptide epitope.In one embodiment, the peptide-containing conjugation partner consistsof a peptide. In one embodiment, the peptide-containing conjugationpartner consists of a peptide comprising a peptide epitope.

In some embodiments, the amino acid-comprising conjugation partnercomprises an epitope bound to the or an amino acid of the conjugationpartner. In some embodiments, the peptide-containing conjugation partnercomprises an epitope bound to the peptide of the peptide-containingconjugation partner. In some embodiments, the epitope is bound to thepeptide via linker group.

In some embodiments, the amino acid-comprising conjugation partnercomprises a peptide epitope bound to the or an amino acid of theconjugation partner via a linker group. In some embodiments, thepeptide-containing conjugation partner comprises a peptide epitope boundto the peptide via a linker group.

In some embodiments, the amino acid-comprising conjugation partnerand/or the peptide-containing conjugation partner comprises an antigenicpeptide. In some embodiments, the peptide conjugate comprises anantigenic peptide.

In some embodiments, the method further comprises coupling the aminoacid of the amino acid conjugate to an amino acid or a peptide toprovide a peptide conjugate, wherein the peptide conjugate comprises oneor more EBV LMP2 epitopes.

In some embodiments, coupling a peptide comprises individually couplingone or more amino acids and/or one or more peptides.

In some embodiments, the method further comprises coupling the aminoacid of the amino acid conjugate or an amino acid of the peptideconjugate to an amino acid or a peptide so as to provide a peptideconjugate comprising a linker group or one or more amino acids thereof.

In some embodiments, the method further comprises coupling an amino acidof the peptide conjugate comprising a linker group or one or more aminoacids thereof to an amino acid or a peptide so as to provide a peptideconjugate comprising one or more EBV LMP2 epitopes bound to the aminoacid to which lipid-containing conjugation partner is conjugated via alinker group.

In some embodiments, the amino acid of the peptide conjugate to whichthe lipid-containing conjugate is conjugated is an N-terminal amino acidresidue.

In some embodiments, the method further comprises coupling the aminoacid of the amino acid conjugate or an amino acid of the peptideconjugate to an amino acid or a peptide so as to provide a peptideconjugate comprising one or more EBV LMP2 epitopes.

In some embodiments, the method further comprises coupling one or moreEBV LMP2 epitopes to the amino acid of the amino acid conjugate or anamino acid of the peptide conjugate. In some embodiments, the methodfurther comprises coupling one or more EBV LMP2 epitopes to the aminoacid of the amino acid conjugate or an amino acid of the peptideconjugate. In some embodiments, the epitope is coupled or bound via alinker group.

In some embodiments, the method further comprises coupling an epitope tothe peptide of the peptide conjugate. In some embodiments, the methodfurther comprises coupling a peptide epitope to the peptide of thepeptide conjugate. In some embodiments, the epitope is bound to thepeptide via a linker group. In various examples, the epitope is a EBVLMP2 epitope.

In one embodiment, the amino acid-comprising conjugation partnerconsists of an amino acid. In one embodiment, the carboxyl group of theC-terminus of the amino acid is protected with a carboxyl protectinggroup and/or the No-amino group of the amino acid is protected with anamino protecting group.

In some embodiments, the carboxyl group of the C-terminus of the peptideis protected with a carboxyl protecting group and/or the No-amino groupof the peptide is protected with an amino protecting group.

In one embodiment, the lipid-containing conjugation partner comprisesone or more optionally substituted straight or branched aliphatic orheteroaliphatic chains each containing at least 4 chain-linked atoms. Inone embodiment, the lipid-containing conjugation partner comprises oneor more optionally substituted straight or branched aliphatic orheteroaliphatic chains each containing at least 6 chain-linked atoms. Inone specifically contemplated embodiment, the one or more chains arealiphatic. In one specifically contemplated embodiment, the one or morechains are saturated.

In some embodiments, the one or more chains are optionally substituted.In some embodiments, the one or more chains are optionally substitutedwith one or more aryl groups.

In some embodiments, the one or more chains comprise at least 4, 6, 8,10, 12, or 14 chain-linked atoms. In some embodiments, the one or morechains comprise from 4-22, 6-22, 8-22, 10-22, 12-22, or 14-22chain-linked atoms.

In one embodiment, the one or more chains are covalently bound to amoiety comprising the carbon-carbon double bond or the thiol by aheteroatom-containing functional group. Examples ofheteroatom-containing functional groups include but are not limited toether, amine, sulfide, sulfoxide, sulfone, ester, amide, carbonate,carbamate, and urea groups.

In exemplary embodiments, the one or more chains are covalently bound tothe moiety by ester functional groups.

In one embodiment, the lipid-containing conjugation partner comprisesone or more saturated or unsaturated fatty acid esters. In someembodiments, the fatty acid is saturated. In one embodiment, one or morefatty acid ester is bound to the moiety comprising to carbon-carbondouble bond or thiol. In one embodiment, the ester is an ester of thecarboxyl group of the fatty acid and an alcohol of the moiety.

In one embodiment, the fatty acid is a C4-22 fatty acid. In oneembodiment, the fatty acid is a C6-22 fatty acid. In another embodiment,the fatty acid is a C10-22 fatty acid. In yet another embodiment, thefatty acid is a C12-22 fatty acid. In one exemplary embodiment, thefatty acid is a C12, C14, C16, C18, or C20 fatty acid.

In some embodiments, the fatty acid is lauric acid, myristic acid,palmitic acid, stearic acid, arachic acid, palmitoleic acid, oleic acid,elaidic acid, linoleic acid, α-linolenic acid, and arachidonic acid. Inone embodiment, the fatty acid is lauric acid, myristic acid, palmiticacid, or stearic acid. In a specifically contemplated embodiment, thefatty acid is palmitic acid.

In one exemplary embodiment, the lipid-containing conjugation partnercomprises one or two fatty acid esters. In a specifically contemplatedembodiment, the lipid-containing conjugation partner comprises one fattyacid ester.

In certain embodiments, the fatty acid ester is an ester of an alcoholcomprising the carbon-carbon double bond or thiol. In one embodiment,the alcohol is a monohydric, dihydric, or trihydric C2-6 aliphaticalcohol. In another embodiment, the alcohol is a monohydric or dihydricC2-4 aliphatic alcohol. In one exemplary embodiment, the alcohol is amonohydric C2 aliphatic or monohydric or dihydric C3 aliphatic alcohol.In a specifically contemplated embodiment, the alcohol is a monohydricC2 alcohol.

In certain embodiments, the lipid-containing conjugation partnercomprises the carbon-carbon double bond.

In one exemplary embodiment, the alcohol comprises the carbon-carbondouble bond.

In a specifically contemplated embodiment, the alcohol is vinyl alcohol.

In specifically contemplated embodiments, the peptide is a syntheticpeptide.

In one embodiment, the amino acid-comprising conjugation partner and/orpeptide conjugate comprises a synthetic peptide. In some embodiments,the synthetic peptide is a peptide prepared by a method comprising solidphase peptide synthesis (SPPS).

In some embodiments, the or an amino acid of the amino acid-comprisingconjugation partner comprises the carbon-carbon double bond or thiol. Insome embodiments, an amino acid residue of the peptide of thepeptide-containing conjugation partner comprises the carbon-carbondouble bond or thiol.

In some embodiments, the amino acid residue comprising the carbon-carbondouble bond or thiol is a terminal amino acid residue. In someembodiments, the terminal amino acid residue is an N-terminal residue.

In some embodiments, the No-amino group of the amino acid comprising thecarbon-carbon double bond or thiol is acylated.

In certain embodiments, the method further comprises acylating theNo-amino group of the amino acid of the amino acid conjugate or theamino acid residue of the peptide conjugate to which thelipid-containing conjugation partner is conjugated. In certainembodiments, the method further comprises acylating the No-amino groupwith a C2-20 fatty acid.

In certain embodiments, the or an amino acid of the aminoacid-comprising conjugation partner comprises the thiol. In certainembodiments, an amino acid residue of the peptide of thepeptide-containing conjugation partner comprises the thiol. In certainembodiments, the thiol is the thiol of a cysteine residue.

In certain embodiments, the cysteine residue is a terminal residue. Incertain embodiments, the cysteine residue is an N-terminal residue.

In some embodiments, the amino group of the cysteine residue isacylated.

In one embodiment, the amino group is acylated with a C2-20 fatty acid.

In one exemplary embodiment, the C2-20 fatty acid is acetyl orpalmitoyl. In another exemplary embodiment, the C2-20 fatty acid isacetyl.

In some embodiments, the amino acid-comprising conjugation partnerand/or peptide conjugate comprises from 8 to 220, 8 to 200, 8 to 175, 8to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50,8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids. In someembodiments, the peptide-containing conjugation partner comprises from 8to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8to 15 amino acids.

In one exemplary embodiment, the amino acid-comprising conjugationpartner and/or peptide conjugate comprises a peptide comprising from 8to 60 amino acids. In one exemplary embodiment, the peptide comprisesfrom 8 to 60 amino acids.

In other embodiments, the amino acid-comprising conjugation partnerand/or peptide conjugate comprises from 5 to 220, 8 to 220, 5 to 175, 8to 175, 8 to 150, 10 to 150, 15 to 125, 20 to 100, 20 to 80, 20 to 60,25 to 100, 25 to 80, 25 to 60, 30 to 80, 40 to 60, or 50 to 60 aminoacids. In other embodiments, the peptide-containing conjugation partnercomprises from 5 to 220, 8 to 220, 5 to 175, 8 to 175, 8 to 150, 10 to150, 15 to 125, 20 to 100, 20 to 80, 20 to 60, 25 to 100, 25 to 80, 25to 60, 30 to 80, 40 to 60, or 50 to 60 amino acids.

In other embodiments, the amino acid-comprising conjugation partnerand/or peptide conjugate comprises from 5 to 150, 5 to 125, 5 to 100, 5to 75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25,or 8 to 20 amino acids. In other embodiments, the peptide-containingconjugation partner comprises from 5 to 150, 5 to 125, 5 to 100, 5 to75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or8 to 20 amino acids.

In one embodiment, the amino acid-comprising conjugation partner and/orpeptide conjugate comprises one or more solubilising groups. In oneembodiment, the peptide-containing conjugation partner comprises one ormore solubilising groups.

In certain embodiments, the solubilising group is an amino acid sequencecomprising two or more hydrophilic amino acid residues in the peptidechain. In certain embodiments, the solubilising group is an amino acidsequence comprising a sequence of two or more consecutive hydrophilicamino acid residues in the peptide chain. In one embodiment, thehydrophilic amino acid residues are cationic amino acid residues. In oneembodiment, the cationic amino acid residues are arginine or lysineresidues. In one specifically contemplated embodiment, the cationicamino acid residues are lysine residues. In one embodiment, the sequencecomprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino acids.In one embodiment, the solubilising group is a tri-, tetra-, penta-,hexa-, or hepta-lysine sequence. In one specifically contemplatedembodiment, the solubilising group is a tetralysine sequence.

In some embodiments, the peptide conjugate and/or amino-acid comprisingconjugation partner comprises a serine residue adjacent to the aminoacid residue to which the lipid-containing conjugation partner isconjugated. In a specifically contemplated embodiment, the peptide ofthe peptide-containing conjugation partner comprises a serine residueadjacent to the amino acid residue to which the lipid-containingconjugation partner is conjugated. In an exemplary embodiment, the aminoacid residue to which the lipid-containing conjugation partner isconjugated is N-terminal. In a specifically contemplated embodiment, thepeptide further comprises a consecutive sequence of two or morehydrophilic amino acid residues adjacent to the serine residue.

In certain embodiments, the peptide conjugate and/or amino-acidcomprising conjugation partner comprises a consecutive sequence of twoor more hydrophilic amino acid residues adjacent to the serine residue.

In certain embodiments, the peptide conjugate and/or aminoacid-comprising conjugation partner comprises only naturally occurringamino acids. In certain embodiments, the peptide-containing conjugationpartner comprises only naturally occurring amino acids. In otherembodiments, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, or 99% or more of the amino acid residues in thepeptide are naturally occurring amino acids.

In other embodiments, 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 97% or more, or 99% or more of the amino acidresidues in the peptide conjugate and/or amino acid-comprisingconjugation partner are naturally occurring amino acids.

In exemplary embodiments, the peptide conjugate and/or aminoacid-comprising conjugation partner comprises a peptide comprising anEBV LMP2 epitope. In exemplary embodiments, the peptide of thepeptide-containing conjugation partner comprises one or more EBV LMP2epitopes.

In various embodiments, the peptide comprises, consists of, or consistsessentially of an amino acid sequence selected from the group consistingof

-   (a) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 1], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is absent    or is a hydrophilic amino acid, Xaa₃ is absent or is a hydrophilic    amino acid, and Xaa₄ is absent or is one or more hydrophilic amino    acids,-   (b) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:2], wherein Xaa1    is absent or is S or a hydrophilic amino acid, Xaa₂ is absent or is    a hydrophilic amino acid, and Xaa₃ is absent or is from one to ten    hydrophilic amino acids,-   (c) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:3], wherein Xaa1 is    absent or is S or a hydrophilic amino acid, and Xaa₂ is absent or is    from one to four hydrophilic amino acids,-   (d) 8 or more contiguous amino acid residues from the sequence    SKKKKDRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:4],-   (e) 8 or more contiguous amino acid residues from the sequence    DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:5],-   (f) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:6],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is    absent or is a hydrophilic amino acid, Xaa₃ is absent or is a    hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (g) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:7], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is absent    or is a hydrophilic amino acid, and Xaa₃ is absent or is from one to    ten hydrophilic amino acids,-   (h) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:8], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, and Xaa₂ is    absent or is from one to four hydrophilic amino acids,-   (i) 8 or more contiguous amino acid residues from the sequence    SKKKKSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:9],-   (j) 8 or more contiguous amino acid residues from the sequence    SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 10],-   (k) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:11], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is absent    or is a hydrophilic amino acid, Xaa₃ is absent or is a hydrophilic    amino acid, and Xaa₄ is absent or is one or more hydrophilic amino    acids,-   (l) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 12], wherein Xaa₁    is absent or is S or a hydrophilic amino acid, Xaa₂ is absent or is    a hydrophilic amino acid, and Xaa₃ is absent or is from one to ten    hydrophilic amino acids,-   (m) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 13], wherein Xaa₁ is    absent or is S or a hydrophilic amino acid, and Xaa₂ is absent or is    from one to four hydrophilic amino acids,-   (n) 8 or more contiguous amino acid residues from the sequence    SKKKKSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 14],-   (o) 8 or more contiguous amino acid residues from the sequence    SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 15],-   (p) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ    ID NO:16], wherein Xaa₁ is absent or is S or a hydrophilic amino    acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is absent    or is a hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (q) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID    NO:17], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is absent or    is from one to ten hydrophilic amino acids,-   (r) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID    NO:18], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    and Xaa₂ is absent or is from one to four hydrophilic amino acids,-   (s) 8 or more contiguous amino acid residues from the sequence    SKKKKDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:19],-   (t) 8 or more contiguous amino acid residues from the sequence    DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:20],-   (u) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO:21],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is    absent or is a hydrophilic amino acid, Xaa₃ is absent or is a    hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (v) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO:22],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is    absent or is a hydrophilic amino acid, and Xaa₃ is absent or is from    one to ten hydrophilic amino acids,-   (w) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO:23], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, and Xaa₂ is    absent or is from one to four hydrophilic amino acids,-   (x) 8 or more contiguous amino acid residues from the sequence    SKKKKLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO:24],-   (y) 8 or more contiguous amino acid residues from the sequence    LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO:25],-   (z) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID    NO:26], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is absent or is    a hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (aa) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:27],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is    absent or is a hydrophilic amino acid, and Xaa₃ is absent or is from    one to ten hydrophilic amino acids,-   (bb) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:28],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, and Xaa₂    is absent or is from one to four hydrophilic amino acids,-   (cc) 8 or more contiguous amino acid residues from the sequence    SKKKKLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:29],-   (dd) 8 or more contiguous amino acid residues from the sequence    LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:30],-   (ee) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:31], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is absent    or is a hydrophilic amino acid, Xaa₃ is absent or is a hydrophilic    amino acid, and Xaa₄ is absent or is one or more hydrophilic amino    acids,-   (ff) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:32], wherein Xaa₁    is absent or is S or a hydrophilic amino acid, Xaa₂ is absent or is    a hydrophilic amino acid, and Xaa₃ is absent or is from one to ten    hydrophilic amino acids,-   (gg) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:33], wherein Xaa₁ is    absent or is S or a hydrophilic amino acid, and Xaa₂ is absent or is    from one to four hydrophilic amino acids,-   (hh) 8 or more contiguous amino acid residues from the sequence    SKKKKLMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:34],-   (ii) 8 or more contiguous amino acid residues from the sequence    LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:35],-   (jj) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:36],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is    absent or is a hydrophilic amino acid, Xaa₃ is absent or is a    hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (kk) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:37], wherein    Xaa₁ is absent or is S or a hydrophilic amino acid, Xaa₂ is absent    or is a hydrophilic amino acid, and Xaa₃ is absent or is from one to    ten hydrophilic amino acids,-   (ll) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:38], wherein Xaa₁    is absent or is S or a hydrophilic amino acid, and Xaa₂ is absent or    is from one to four hydrophilic amino acids,-   (mm) 8 or more contiguous amino acid residues from the sequence    SKKKKLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:39],-   (nn) 8 or more contiguous amino acid residues from the sequence    LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:40],-   (oo) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIA    GGSI [SEQ ID NO:41], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is    absent or is a hydrophilic amino acid, and Xaa₄ is absent or is one    or more hydrophilic amino acids,-   (pp) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI    [SEQ ID NO:42], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃    is absent or is from one to ten hydrophilic amino acids,-   (qq) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI    [SEQ ID NO:43], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, and Xaa₂ is absent or is from one to four hydrophilic    amino acids,-   (rr) 8 or more contiguous amino acid residues from the sequence    SKKKKLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI [SEQ    ID NO:44],-   (ss) 8 or more contiguous amino acid residues from the sequence    LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI [SEQ ID    NO:45],-   (tt) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID    NO:46], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is absent or is    a hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (uu) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID    NO:47], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is absent or    is from one to ten hydrophilic amino acids,-   (vv) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO:48],    wherein Xaa₁ is absent or is S or a hydrophilic amino acid, and Xaa₂    is absent or is from one to four hydrophilic amino acids,-   (ww) 8 or more contiguous amino acid residues from the sequence    SKKKKFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO:49],-   (xx) 8 or more contiguous amino acid residues from the sequence    FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO:50],-   (yy) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL    [SEQ ID NO:51], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is    absent or is a hydrophilic amino acid, and Xaa₄ is absent or is one    or more hydrophilic amino acids,-   (zz) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ    ID NO:52], wherein Xaa₁ is absent or is S or a hydrophilic amino    acid, Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is    absent or is from one to ten hydrophilic amino acids,-   (aaa) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID    NO:53], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    and Xaa₂ is absent or is from one to four hydrophilic amino acids,-   (bbb) 8 or more contiguous amino acid residues from the sequence    SKKKKLQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID    NO:54],-   (ccc) 8 or more contiguous amino acid residues from the sequence    LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID NO:55],-   (ddd) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLI    FLIGFA [SEQ ID NO:56], wherein Xaa₁ is absent or is S or a    hydrophilic amino acid, Xaa₂ is absent or is a hydrophilic amino    acid, Xaa₃ is absent or is a hydrophilic amino acid, and Xaa₄ is    absent or is one or more hydrophilic amino acids,-   (eee) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIG    FA [SEQ ID NO:57], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃    is absent or is from one to ten hydrophilic amino acids,-   (fff) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFA    [SEQ ID NO:58], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, and Xaa₂ is absent or is from one to four hydrophilic    amino acids,-   (ggg) 8 or more contiguous amino acid residues from the sequence    SKKKKSGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFA [SEQ    ID NO:59],-   (hhh) 8 or more contiguous amino acid residues from the sequence    SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFA [SEQ ID    NO:60],-   (iii) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ    ID NO:61], wherein Xaa₁ is absent or is S or a hydrophilic amino    acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is absent    or is a hydrophilic amino acid, and Xaa₄ is absent or is one or more    hydrophilic amino acids,-   (jjj) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID    NO:62], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is absent or    is from one to ten hydrophilic amino acids,-   (kkk) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID    NO:63], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    and Xaa₂ is absent or is from one to four hydrophilic amino acids,-   (lll) 8 or more contiguous amino acid residues from the sequence    SKKKKSNEEPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID NO:64],-   (mmm) 8 or more contiguous amino acid residues from the sequence    SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID NO:65],-   (nnn) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAA S    [SEQ ID NO:66], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is    absent or is a hydrophilic amino acid, and Xaa₄ is absent or is one    or more hydrophilic amino acids,-   (ooo) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ    ID NO:67], wherein Xaa₁ is absent or is S or a hydrophilic amino    acid, Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is    absent or is from one to ten hydrophilic amino acids,-   (ppp) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID    NO:68], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    and Xaa₂ is absent or is from one to four hydrophilic amino acids,-   (qqq) 8 or more contiguous amino acid residues from the sequence    SKKKKGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID    NO:69],-   (rrr) 8 or more contiguous amino acid residues from the sequence    GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID NO:70],-   (sss) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃Xaa₄AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT    [SEQ ID NO:71], wherein Xaa₁ is absent or is S or a hydrophilic    amino acid, Xaa₂ is absent or is a hydrophilic amino acid, Xaa₃ is    absent or is a hydrophilic amino acid, and Xaa₄ is absent or is one    or more hydrophilic amino acids,-   (ttt) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂Xaa₃AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ    ID NO:72], wherein Xaa₁ is absent or is S or a hydrophilic amino    acid, Xaa₂ is absent or is a hydrophilic amino acid, and Xaa₃ is    absent or is from one to ten hydrophilic amino acids,-   (uuu) 8 or more contiguous amino acid residues from the sequence    Xaa₁Xaa₂AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID    NO:73], wherein Xaa₁ is absent or is S or a hydrophilic amino acid,    and Xaa₂ is absent or is from one to four hydrophilic amino acids,-   (vvv) 8 or more contiguous amino acid residues from the sequence    SKKKKAAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID    NO:74],-   (www) 8 or more contiguous amino acid residues from the sequence    AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO:75],-   (xxx) the sequence of any one of SEQ ID NOs: 1 to 75,-   (yyy) 8 or more contiguous amino acid residues from the sequence of    any one of    -   ESNEEPPPPY [SEQ ID NO: 76],    -   SNEEPPPPY [SEQ ID NO: 77],    -   HSDYQPLGT [SEQ ID NO: 78],    -   PLGTQDQSL [SEQ ID NO: 79],    -   PLGTQDQSLY [SEQ ID NO: 80],    -   PLGTQDQSLY [SEQ ID NO: 80],    -   LGTQDQSLY [SEQ ID NO: 81],    -   GTQDQSLYL [SEQ ID NO: 82],    -   GTQDQSLYL [SEQ ID NO: 83],    -   GTQDQSLYLG [SEQ ID NO: 84],    -   QSLYLGLQH [SEQ ID NO: 85],    -   SLYLGLQHD [SEQ ID NO: 86],    -   SLYLGLQHD [SEQ ID NO: 86],    -   GLQHDGNDGL [SEQ ID NO: 87],    -   GNDGLPPPPY [SEQ ID NO: 88],    -   GLPPPPYSP [SEQ ID NO: 89],    -   GLPPPPYSPR [SEQ ID NO: 90],    -   GLPPPPYSPR [SEQ ID NO: 90],    -   PRDDSSQHIY [SEQ ID NO: 91],    -   RDDSSQHIY [SEQ ID NO: 92],    -   HIYEEAGRG [SEQ ID NO: 93],    -   ILLARLFLY [SEQ ID NO: 94],    -   SSCSSCPLSKI [SEQ ID NO: 95],    -   LLWTLVVLL [SEQ ID NO: 96],    -   FLYALALLL [SEQ ID NO: 97],    -   CLGGLLTMV [SEQ ID NO: 98],    -   LIVDAVLQL [SEQ ID NO: 99],    -   LTAGFLIFL [SEQ ID NO: 100],    -   TVCGGIMFL [SEQ ID NO: 101],-   (zzz) the sequence of any one of SEQ ID NOs: 76-101,-   (aaaa) or any combination of two or more of (a) to (zzz) above.

In one exemplary embodiment, the peptide comprises one or more epitopesderived from Latent Membrane Protein 2 (LMP2), for example, fromfull-length EBV LMP2 (amino acids 1-497). In one specificallycontemplated embodiment, the peptide comprises, consists essentially of,or consists of an amino acid sequence selected from the group consistingof 8 or more contiguous amino acid residues from any one of SEQ ID NOs:4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29, 30, 34, 35, 39, 40, 44, 45, 49,50, 54, 55, 59, 60, 64, 65, 69, 70, 74, or 75.

In another specifically contemplated embodiment, the peptide comprises,consists essentially of, or consists of an amino acid sequence selectedfrom the group consisting of 12 or more contiguous amino acid residuesfrom any one of SEQ ID NOs: 4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29, 30,34, 35, 39, 40, 44, 45, 49, 50, 54, 55, 59, 60, 64, 65, 69, 70, 74, or75.

In another specifically contemplated embodiment, the peptide comprises,consists essentially of, or consists of an amino acid sequence selectedfrom the group consisting of 15 or more, 18 or more, 20 or more, or 25or more contiguous amino acid residues from any one of SEQ ID NOs: 4, 5,9, 10, 14, 15, 19, 20, 24, 25, 29, 30, 34, 35, 39, 40, 44, 45, 49, 50,54, 55, 59, 60, 64, 65, 69, 70, 74, or 75.

In one embodiment, the peptide comprises, consists essentially of, orconsists of an amino acid sequence selected from the group consisting ofany one of SEQ ID NOs: 4, 5, 9, 10, 14, 15, 19, 20, 24, 25, 29, 30, 34,35, 39, 40, 44, 45, 49, 50, 54, 55, 59, 60, 64, 65, 69, 70, 74, or 75.

In another specifically contemplated embodiment, the peptide comprises,consists essentially of, or consists of an amino acid sequence selectedfrom the group consisting of 15 or more, 18 or more, 20 or more, or 25or more contiguous amino acid residues from any one of SEQ ID NOs: 1 to75.

In one embodiment, the peptide comprises, consists essentially of, orconsists of an amino acid sequence selected from the group consisting ofany one of SEQ ID NOs: 1 to 75.

In one embodiment, the peptide comprises an amino acid sequence selectedfrom the group consisting of any one of SEQ ID NOs: 76 to 101. In oneexample, the peptide comprises an amino acid sequence selected from thegroup consisting of any one of SEQ ID NOs: 76 to 93.

In one embodiment, the peptide comprises an amino acid sequence selectedfrom the group consisting of any two or more of SEQ ID NOs: 76 to 101.In one example, the peptide comprises an amino acid sequence selectedfrom the group consisting of any two or more of SEQ ID NOs: 76 to 93.

In one specifically contemplated embodiment, the reactive functionalgroups of the amino acids of the peptide-containing conjugation partnerare unprotected.

In certain embodiments, one or more reactive functional groups of one ormore amino acids of the peptide conjugate are unprotected.

In certain embodiments, one or more reactive functional groups of theamino acid of the amino acid conjugate are unprotected.

In certain embodiments, one or more reactive functional groups of one ormore amino acids of the amino acid-comprising conjugation partner areunprotected.

In certain embodiments, the amino acid-comprising conjugation partnercomprises a peptide, wherein the reactive functional groups of the sidechains of the amino acids of the peptide are unprotected, with theexception of any thiols other than the thiol to be reacted.

In one specifically contemplated embodiment, the reactive functionalgroups of the amino acids of the peptide of the peptide-containingconjugation partner are unprotected. In one specifically contemplatedembodiment, the reactive functional groups of the amino acids of thepeptide of the peptide-containing conjugation partner are unprotected,with the exception of any thiols other than the thiol to be reacted.

In one aspect, the invention relates to a method of making apeptide-conjugate comprising a structure of the formula (A):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R1 is L2-C(O)—OC1-6alkyl;    -   R3, R4, R5, R8, and R9 are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R3 is L2-C(O)—OC1-6alkyl;    -   or R9 is an amino protecting group, L3-C(O), or A2;    -   R6 and R7 at each instance of n are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R3 is L2-C(O)—OC1-6alkyl, R1 is not L2-C(O)—OC1-6alkyl;            and        -   when m is an integer from 2 to 4, no more than one R1 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl or heteroalkyl present in any of        R1, R2, R3, R4, R5, R6, R7, R8, R9, L1, L2 and L3 is optionally        substituted;    -   or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the method comprises making a peptide-conjugatecomprising a structure of the formula (A):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R1 is L2-C(O)—OC1-6alkyl;    -   R3, R4, R5, R8, and R9 are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R3 is L2-C(O)—OC1-6alkyl;    -   or R9 is L3-C(O) or A2;    -   R6 and R7 at each instance of n are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R9 is not A2, A1 is a peptide;        -   when R3 is L2-C(O)—OC1-6alkyl, R1 is not L2-C(O)—OC1-6alkyl;            and        -   when m is an integer from 2 to 4, no more than one R1 is            L2-C(O)—OC1-6alkyl; and wherein any alkyl, cycloalkyl or            heteroalkyl present in any of R1, R2, R3, R4, R5, R6, R7,            R8, R9, L1, L2 and L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the method comprises making an amino acid orpeptide-conjugate comprising a structure of the formula (B):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   p is an integer from 0 to 4;    -   q is an integer from 0 to 2;    -   R11 and R22 at each instance of p are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is        L2-C(O)—OC1-6alkyl;    -   R33, R44, R55, R66, R77, R8, and R9 are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is        L2-C(O)—OC1-6alkyl;    -   or R9 is an amino protecting group, L3-C(O), or A2;    -   Ra and Rb at each instance of q are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R33 is L2-C(O)—OC1-6alkyl, R11 is not            L2-C(O)—OC1-6alkyl; and        -   when p is an integer from 2 to 4, no more than one R11 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl, or heteroalkyl present in any of        R11, R22, R 33, R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and        L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the method comprises making a peptide-conjugatecomprising a structure of the formula (B):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   p is an integer from 0 to 4;    -   q is an integer from 0 to 2;    -   R11 and R22 at each instance of p are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is        L2-C(O)—OC1-6alkyl;    -   R33, R44, R55, R66, R77, R8, and R9 are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is        L2-C(O)—OC1-6alkyl;    -   or R9 is L3-C(O) or A2;    -   Ra and Rb at each instance of q are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R9 is not A2, A1 is a peptide;        -   when R33 is L2-C(O)—OC1-6alkyl, R11 is not            L2-C(O)—OC1-6alkyl; and        -   when p is an integer from 2 to 4, no more than one R11 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl, or heteroalkyl present in any of        R11, R22, R 33, R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and        L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the lipid-containing conjugation partner is acompound of the formula (A1):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   m is an integer from 0 to 4;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R1 is L2-C(O)—OC1-6alkyl; R3,        R4, and R5 are each independently hydrogen, C1-6alkyl, or        C3-6cycloalkyl; or R3 is L2-C(O)—OC1-6alkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   provided that:        -   when R3 is L2-C(O)—OC1-6alkyl, R1 is not L2-C(O)—OC1-6alkyl;            and        -   when m is an integer from 2 to 4, no more than one R1 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl or heteroalkyl present in any of        R1, R2, R3, R4, R5, L1, and L2 is optionally substituted,    -   or a pharmaceutically acceptable salt or solvate thereof.

In another embodiment, the lipid containing conjugation partner is acompound of the formula (B1):

wherein

-   -   Z is selected from the group consisting of —O—, —NR—, —S—,        —S(O)—, —SO₂—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, —OC(O)O—,        —NRC(O)O—, —OC(O)NR—, and —NRC(O)NR—;    -   R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the alkyl        or cycloalkyl is optionally substituted;    -   p is an integer from 0 to 4;    -   R11 and R22 at each instance of p are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is        L2-C(O)—OC1-6alkyl;    -   R33 and R44 are each independently hydrogen, C1-6alkyl, or        C3-6cycloalkyl; or R33 is L2-C(O)—OC1-6alkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   provided that:        -   when R33 is L2-C(O)—OC1-6alkyl, R11 is not            L2-C(O)—OC1-6alkyl; and        -   when p is an integer from 2 to 4, no more than one R11 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl, or heteroalkyl present in any of        R11, R22, R 33, R44, L1, and L2 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the lipid-containing conjugation partner is acompound of the formula (II) as defined in any of the embodimentsdescribed herein.

In one embodiment, the lipid-containing conjugation partner is acompound of the formula (IIA) as defined in any of the embodimentsdescribed herein.

In one embodiment, the amino acid-comprising conjugation partner is acompound of the formula (III) as defined in any of the embodimentsdescribed herein.

In one embodiment, the peptide-containing conjugation partner is acompound of the formula (III) as defined in any of the embodimentsdescribed herein.

In one embodiment, the amino acid-comprising conjugation partner is acompound of the formula (IIIA) as defined in any of the embodimentsdescribed herein.

In one embodiment, the peptide-containing conjugation partner is acompound of the formula (IIIA) as defined in any of the embodimentsdescribed herein.

In one embodiment, the method comprises making an amino acid or peptideconjugate comprising a structure of the formula (I)

wherein

-   -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R1 is L2-C(O)—OC1-6alkyl; R3,        R4, R5, R8, and R9 are each independently hydrogen, C1-6alkyl,        or C3-6cycloalkyl; or R3 is L2-C(O)—OC1-6alkyl;    -   or R9 is an amino protecting group, L3-C(O), or A2; R6 and R7 at        each instance of n are each independently hydrogen, C1-6alkyl,        or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R3 is L2-C(O)—OC1-6alkyl, R1 is not L2-C(O)—OC1-6alkyl;            and        -   when m is an integer from 2 to 4, no more than one R1 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl or heteroalkyl present in any of        R1, R2, R3, R4, R5, R6, R7, R8, R9, L1, L2 and L3 is optionally        substituted;    -   or a pharmaceutically acceptable salt or solvate thereof;

the method comprising reacting a lipid-containing conjugation partner ofthe formula (II)

wherein m, R1, R2, R3, R4, R5, and L1 are as defined in the compound offormula (I);

and a peptide-containing conjugation partner comprising a structure ofthe formula (III)

wherein n, R6, R7, R8, R9 and A1 are as defined in the compound offormula (I);

under conditions effective to conjugate the compound of formula (II)with the compound of formula (III) by hydrothiolation of thecarbon-carbon double bond in the compound of formula (II) with the thiolin the compound of formula (III).

In one embodiment, the method comprises making a peptide conjugatecomprising a structure of the formula (I)

wherein

-   -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R1 is L2-C(O)—OC1-6alkyl;    -   R3, R4, R5, R8, and R9 are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R3 is L2-C(O)—OC1-6alkyl;    -   or R9 is L3-C(O) or A2;    -   R6 and R7 at each instance of n are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R9 is not A2, A1 is a peptide;        -   when R3 is L2-C(O)—OC1-6alkyl, R1 is not L2-C(O)—OC1-6alkyl;            and when m is an integer from 2 to 4, no more than one R1 is            L2-C(O)—OC1-6alkyl; and wherein any alkyl, cycloalkyl or            heteroalkyl present in any of R1, R2, R3, R4, R5, R6, R7,            R8, R9, L1, L2 and L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof;

the method comprising reacting a lipid-containing conjugation partner ofthe formula (II)

wherein m, R1, R2, R3, R4, R5, and L1 are as defined in the compound offormula (I);

and a peptide-containing conjugation partner comprising a structure ofthe formula (III)

wherein n, R6, R7, R8, R9 and A1 are as defined in the compound offormula (I);

under conditions effective to conjugate the compound of formula (II)with the compound of formula (III) by hydrothiolation of thecarbon-carbon double bond in the compound of formula (II) with the thiolin the compound of formula (III).

In one embodiment, the method comprises making an amino acid or peptideconjugate comprising a structure of the formula (IA),

wherein

-   -   p is an integer from 0 to 4;    -   q is an integer from 0 to 2;    -   R11 and R22 at each instance of p are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is        L2-C(O)—OC1-6alkyl;    -   R33, R44, R55, R66, R77, R8, and R9 are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is        L2-C(O)—OC1-6alkyl;    -   or R9 is an amino protecting group, L3-C(O), or A2;    -   Ra and Rb at each instance of q are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R33 is L2-C(O)—OC1-6alkyl, R11 is not            L2-C(O)—OC1-6alkyl; and        -   when p is an integer from 2 to 4, no more than one R11 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl, or heteroalkyl present in any of        R11, R22, R 33, R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and        L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof;

the method comprising reacting a compound of the formula (IIA)

wherein p, R11, R22, R33, R44, and L1 are as defined in the compound offormula (IA);

and a compound of the formula (IIIA)

wherein q, R55, R66, R77, R8, R9, Ra, Rb, and A1 are as defined in thecompound of formula (IA);

under conditions effective to conjugate the compound of formula (IIA)with the compound of formula (IIIA) by hydrothiolation of thecarbon-carbon double bond in the compound of formula (IIIA) with thethiol in the compound of formula (IIA).

In one embodiment, the method comprises making a peptide conjugatecomprising a structure of the formula (IA),

wherein

-   -   p is an integer from 0 to 4;    -   q is an integer from 0 to 2;    -   R11 and R22 at each instance of p are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is        L2-C(O)—OC1-6alkyl;    -   R33, R44, R55, R66, R77, R8, and R9 are each independently        hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is        L2-C(O)—OC1-6alkyl;    -   or R9 is L3-C(O) or A2;    -   Ra and Rb at each instance of q are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R9 is not A2, A1 is a peptide;        -   when R33 is L2-C(O)—OC1-6alkyl, R11 is not            L2-C(O)—OC1-6alkyl; and        -   when p is an integer from 2 to 4, no more than one R11 is            L2-C(O)—OC1-6alkyl; and    -   wherein any alkyl, cycloalkyl, or heteroalkyl present in any of        R11, R22, R 33, R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and        L3 is optionally substituted;    -   or a pharmaceutically acceptable salt or solvate thereof;

the method comprising reacting a compound of the formula (IIA)

wherein p, R11, R22, R33, R44, and L1 are as defined in the compound offormula (IA);

and a compound of the formula (IIIA)

wherein q, R55, R66, R77, R8, R9, Ra, Rb, and A1 are as defined in thecompound of formula (IA);

under conditions effective to conjugate the compound of formula (IIA)with the compound of formula (IIIA) by hydrothiolation of thecarbon-carbon double bond in the compound of formula (IIIA) with thethiol in the compound of formula (IIA).

In one embodiment, at least one of L1 and L2 is C5-22alkyl.

In one embodiment, p is an integer from 0 to 2. In another embodiment, pis 0 or 1.

In some embodiments, R11 and R22 at each instance of p are eachindependently hydrogen; or R11 is L2-C(O)—OCH2. In one embodiment, R11on the carbon adjacent to L1-C(O)—O is L2-C(O)—OCH2.

In one specifically contemplated embodiment, R11 and R22 at eachinstance of p are each independently hydrogen.

In one embodiment, R33 is hydrogen or L2-C(O)—OCH2.

In one embodiment, R33 and R44 are each hydrogen.

In one specifically contemplated embodiment, q is 0 or 1. In onespecifically contemplated embodiment, q is 0.

In one specifically contemplated embodiment, R55, R66, and R77 are eachhydrogen.

In some embodiments, Ra and Rb are at each instance of q are eachhydrogen.

In one embodiment, L1 is C11-21alkyl; p is 1; R11 is hydrogen orL2-C(O)—OCH2; R22 is hydrogen; R33 is hydrogen or L2-C(O)—OCH2; R44 ishydrogen; and L2 is C11-21alkyl.

In one embodiment, R55, R66, R77, Ra, Rb and R8 are each hydrogen; andR9 is hydrogen, L3-C(O), or A2. In one embodiment, R55, R66, R77, Ra, Rband R8 are each hydrogen; and R9 is hydrogen or L3-C(O).

In one embodiment, L1 is C11-21alkyl; p is 1; R11 is hydrogen orL2-C(O)—OCH2; R22 is hydrogen; R33 is hydrogen or L2-C(O)—OCH2; R44 ishydrogen; L2 is C11-21alkyl; R55, R66, R77, Ra, Rb and R8 are eachhydrogen; and R9 is hydrogen, L3-C(O), or A2.

In one embodiment, L1 is C5-21alkyl. In another embodiment, L1 isC9-21alkyl. In yet another embodiment, L1 is C11-21alkyl. In oneexemplary embodiment, L1 is C11, C13, C15, C17, or C19alkyl. In onespecifically contemplated embodiment, L1 is C15alkyl.

In one embodiment, L1 comprises a linear chain of 9-21 carbon atoms. Inone specifically contemplated embodiment, L1 is linear C15alkyl.

In one embodiment, m is an integer from 0 to 2. In another embodiment, mis 0 or 1.

In one specifically contemplated embodiment, m is 0.

In some embodiments, R1 and R2 at each instance of m are eachindependently hydrogen; or R1 is L2-C(O)—OCH2. In one embodiment, R1 onthe carbon atom adjacent to L1-C(O)—O is L2-C(O)—OCH2.

In one specifically contemplated embodiment, R1 and R2 at each instanceof m are each independently hydrogen.

In one embodiment, R3 is hydrogen or L2-C(O)—OCH2. In one specificallycontemplated embodiment, R3 is hydrogen.

In one embodiment, L2 is C5-21alkyl. In another embodiment, L2 isC9-21alkyl. In yet another embodiment, L2 is C11-21 alkyl. In oneexemplary embodiment, L2 is C11, C13, C15, C17, or C19alkyl. In anotherexemplary embodiment, L2 is C15alkyl.

In one specifically contemplated embodiment, R4 and R5 are eachhydrogen.

In one specifically contemplated embodiment, n is 1.

In one specifically contemplated embodiment, R6 and R7 are eachhydrogen.

In exemplary embodiments, R8 is hydrogen.

In one embodiment, R8 and R9 are each hydrogen; or R9 is L3-C(O) or A2.In one exemplary embodiment R8 is hydrogen and R9 is L3-C(O).

In some embodiments, L3 is C1-21alkyl. In one specifically contemplatedembodiment, L3 is methyl or linear C15alkyl. In exemplary embodiments,L3 is methyl.

Those skilled in the art will appreciate that the structures of formula(III) and (IIIA) may comprise a peptide of the peptide-containingconjugation partner. As described herein, the peptide may be optionallysubstituted, modified, or bound to various other moieties as describedherein to provide the peptide-containing conjugation partner.

In one embodiment, A1 is a peptide comprising an EBV LMP2 epitope. Inone embodiment A2 is a peptide comprising an EBV LMP2 epitope.

In one embodiment, A1 is a peptide substituted with an epitope. In oneembodiment, A2 is a peptide substituted with an epitope.

In one embodiment, the epitope is bound to the peptide via a linkergroup.

In one embodiment, the epitope is a peptide epitope.

In some embodiments, A1 and/or A2 are each independently a peptidecomprising from about 8 to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125,8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30,8 to 25, 8 to 20, or 8 to 15 amino acids. In one exemplary embodiment,A1 and A2 are each independently a peptide comprising from about 8 to 60amino acids.

In other embodiments, A1 and/or A2 are each independently a peptidecomprising from about 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 60, 5to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to 125, 8 to 100,8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or 8 to 20 aminoacids.

In some embodiments, A1 and/or A2 are each independently a peptide,wherein the peptide comprises 8 to 60 amino acids.

In some embodiments, A1 and/or A2 are each independently a peptidecomprising or substituted with a peptide epitope, wherein the peptideepitope comprises from 8 to 60 amino acids.

In some embodiments, A1 and/or A2 are each independently a peptidecomprising or substituted with a peptide epitope, wherein the peptidecomprises, consists of, or consists essentially of an amino acidsequence selected from the group consisting of 8 or more, 10 or more, 12or more, 15 or more, 20 or more, or 25 or more contiguous amino acidsfrom the sequence of any one of SEQ ID NOs: 1-101, for example from thesequence of any one of SEQ ID NOs: 1-75.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen orL2-C(O)—OCH2; L2 is C11-21alkyl; and R4 and R5 are each hydrogen.

In one embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 ishydrogen, L3-C(O), or A2. In one embodiment, n is 1; R6, R7, and R8 areeach hydrogen; and R9 is hydrogen or L3-C(O). In one embodiment, L3 ismethyl or linear C15alkyl.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen orL2-C(O)—OCH2; L2 is C11-21alkyl; R4 and R5 are each hydrogen; n is 1;R6, R7, and R8 are each hydrogen; R9 is hydrogen, L3-C(O), or A2. In oneembodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen or L2-C(O)—OCH2;L2 is C11-21alkyl; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8are each hydrogen; R9 is hydrogen or L3-C(O).

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; and R4 andR5 are each hydrogen.

In one embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 ishydrogen, L3-C(O), or A2. In one embodiment, n is 1; R6, R7, and R8 areeach hydrogen; and R9 is hydrogen or L3-C(O). In one embodiment, n is 1;R6, R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O), whereinL3 is methyl.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen, L3-C(O), or A2. In one embodiment, L1 is C11-21alkyl; m is 0;R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 areeach hydrogen; R9 is hydrogen or L3-C(O).

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen or L3-C(O), wherein L3 is methyl.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; and R4 andR5 are each hydrogen.

In one embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 ishydrogen, 13-C(O), or A2. In one embodiment, n is 1; R6, R7, and R8 areeach hydrogen; and R9 is hydrogen or L3-C(O). In one embodiment, n is 1;R6, R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O), whereinL3 is methyl.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen, L3-C(O), or A2. In one embodiment, L1 is C11-21alkyl; m is 0;R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 areeach hydrogen; R9 is hydrogen or L3-C(O).

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen or L3-C(O), wherein L3 is methyl.

In some embodiments, A1 is a peptide comprising serine as the firstN-terminal amino acid residue. In some embodiments, A1 and/or A2 is apeptide comprising a solubilising group. In some embodiments, thesolubilising group comprises an amino acid sequence comprising two ormore hydrophilic amino acid residues in the peptide chain. In certainembodiments, A1 is a peptide comprising a solubilising group comprisingan amino acid sequence comprising two or more hydrophilic amino acidresidues in the peptide chain.

In some embodiments, A1 is a peptide comprising serine as the firstN-terminal amino acid residue and a solubilising group comprising anamino acid sequence comprising two or more hydrophilic amino acidresidues in the peptide chain adjacent to the serine.

In some embodiments, the solubilising group comprises an amino acidsequence comprising two or more consecutive hydrophilic amino acidresidues in the peptide chain.

In one embodiment, the hydrophilic amino acid residues are cationicamino acid residues.

In one embodiment, the cationic amino acid residues are arginine orlysine residues. In one specifically contemplated embodiment, thecationic amino acid residues are lysine residues. In one embodiment, thesequence comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5amino acids. In one embodiment, the solubilising group is a tri-,tetra-, penta-, hexa-, or hepta-lysine sequence. In one specificallycontemplated embodiment, the solubilising group is a tetralysinesequence.

In some embodiments, R9 is hydrogen, an amino protecting group orL3-C(O). In some embodiments, R9 is hydrogen or L3-C(O).

In some embodiments, R9 is hydrogen or an amino protecting group, andthe method further comprises acylating the amino acid conjugate orpeptide conjugate so as to replace the hydrogen or amino protectinggroup at R9 with L3-C(O). In some embodiments, acylating the amino acidconjugate or peptide conjugate so as to replace the amino protectinggroup at R9 with L3-C(O) comprises removing the amino protecting groupat R9 to provide a hydrogen at R9.

In some embodiments, A1 and/or A2 is an amino acid or a peptide. In someembodiments, A1 and/or A2 is a peptide.

In some embodiments, A1 is OH or OP1 and/or R9 is hydrogen, an aminoprotecting group or L3-C(O). In some embodiments, A1 is OP1 or OH and/orR9 is hydrogen, an amino protecting group or L3-C(O). In someembodiments, A1 is a OP1 or OH and R9 is hydrogen, an amino protectinggroup or L3-C(O).

In some embodiments, A1 is a OP1 or OH and/or R9 is hydrogen, an aminoprotecting group or L3-C(O), and the method comprises coupling an aminoacid or a peptide so as to replace A1 and/or R9 with the amino acid orpeptide.

In some embodiments, A1 is a OP1 or OH and R9 is hydrogen, an aminoprotecting group or L3-C(O) and the method further comprises coupling anamino acid or a peptide so as to replace A1 and/or R9 with the aminoacid or peptide.

In some embodiments, coupling a peptide comprises individually couplingone or more amino acids and/or one or more peptides.

In some embodiments, coupling the amino acid or peptide provides apeptide conjugate comprising a peptide epitope. In some embodiments, thecoupling the amino acid or peptide provides a peptide conjugatecomprising a linker group or one or more amino acids thereof. In someembodiments, coupling the amino acid or peptide provides a peptideconjugate comprising a peptide epitope bound to the amino acid to whichlipid-containing conjugation partner is conjugated via a linker group.

In some embodiments, the amino protecting group is Boc, Fmoc, Cbz(carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl), Bpoc(2-(4-biphenyl)isopropoxycarbonyl) and Dde(1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl). In some embodiments, theamino protecting group is Boc or Fmoc.

In some embodiments, the carboxyl protecting group is tert-butyl orbenzyl. In one embodiment, the compound of the formula (I) is a compoundof the formula (IV):

wherein

-   -   R3 is hydrogen or L2-C(O)—OCH2;    -   R9 is hydrogen, an amino protecting group, L3-C(O), or A2; and    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;

or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the compound of the formula (I) is a compound of theformula (IV):

wherein

-   -   R3 is hydrogen or L2-C(O)—OCH2;    -   R9 is hydrogen, L3-C(O), or A2; and    -   L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-21alkyl or C4-20heteroalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:    -   when R9 is not A2, A1 is a peptide;

or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, L1, A1, A2, L2, and L3 in the compound of formula(IV) are each independently as defined in any of the embodimentsrelating to the compound of the formula (I).

In one specifically contemplated embodiment, R3 is hydrogen.

In another specifically contemplated embodiment, R9 is acetyl.

In another specifically contemplated embodiment, R3 is hydrogen and R9is acetyl.

In some embodiments, the method is for making a compound of the formula(IV), wherein L1 is C15 linear alkyl, R3 is hydrogen, R9 is Fmoc, and A1is OH, and the method comprises reacting vinyl palmitate andFmoc-Cys-OH.

In some embodiments, the amino protecting group is not Fmoc. In someembodiments, the amino protecting group is Boc.

In some embodiments, the amino acid-comprising conjugation partner isnot Fmoc-Cys-OH.

In some embodiments, the peptide conjugate comprises 3 or more, 4 ormore, or 5 or more contiguous amino acids. In some embodiments, thecompound of formula (I) comprises 3 or more, 4 or more, or 5 or morecontiguous amino acids.

In one embodiment, the conditions effective to conjugate thelipid-containing conjugation partner to the amino acid-comprisingconjugation partner comprises the generation of one or more freeradicals. In one embodiment, the conditions effective to conjugate thelipid-containing conjugation partner to the peptide-containingconjugation partner comprises the generation of one or more freeradicals.

In some embodiments, the generation of one or more free radicals isinitiated thermally and/or photochemically. In certain embodiments, thegeneration of one or more free radicals is initiated by the thermaland/or photochemical degradation of a free radical initiator. Inexemplary embodiments, the generation of one or more free radicals isinitiated by the thermal degradation of a thermal initiator or thephotochemical degradation of a photochemical initiator.

In some embodiments, thermal degradation of the free radical initiatorcomprises heating the reaction mixture at a suitable temperature. Insome embodiments, the reaction mixture is heated at a temperature fromabout 40° C. to about 200° C., from about 50° C. to about 180° C., fromabout 60° C. to about 150° C., from about 65° C. to about 120° C., fromabout 70° C. to about 115° C., from about 75° C. to about 110° C., orfrom about 80° C. to about 100° C. In other embodiments, the reactionmixture is heated at a temperature of at least about 40° C., at leastabout 50° C., at least about 60° C., or at least about 65° C. In onespecifically contemplated embodiment, the reaction mixture is heated ata temperature of about 90° C.

In some embodiments, photochemical degradation of the free radicalinitiator comprises irradiation with ultraviolet light. In aspecifically contemplated embodiment, the ultraviolet light has awavelength of about 365 nm. In exemplary embodiments, photochemicaldegradation of the free radical initiator is carried out at aboutambient temperature.

In one specifically contemplated embodiment, the thermal initiator is2,2′-azobisisobutyronitrile (AIBN). In one specifically contemplatedembodiment, the photoinitiator is 2,2-dimethoxy-2-phenylacetophenone(DMPA).

In certain embodiments, the reaction is carried out in a liquid medium.In one embodiment, the liquid medium comprises a solvent. In oneembodiment, the solvent is selected from the group consisting ofN-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO),N,N-dimethylformamide (DMF), dichloromethane (DCM), 1,2-dichloroethane,and mixtures thereof. In one specifically contemplated embodiment, thesolvent comprises NMP, DMSO, or a mixture thereof.

In one specifically contemplated embodiment, the solvent comprises DMSO.

In some embodiments, the reaction is carried out in the presence of oneor more additives that inhibit dimerisation, telomerisation, orpolymerisation. In some exemplary embodiments, the additive is selectedfrom the group consisting of reduced glutathione (GSH),2,2′-(ethylenedioxy)diethanethiol (DODT), 1,4-dithiothreitol (DTT), andprotein. In a specifically contemplated embodiment, the additive is DTT.In some embodiments, the additive is DTT or tert-butyl mercaptan.

In some embodiments, the one or more additive is selected from the groupconsisting of TFA, tert-butyl mercaptan, and a combination thereof. Incertain embodiments, the one or more additive is a combination of TFAand tert-butyl mercaptan. In some embodiments, the reaction is carriedout for a period of time from about 5 minutes to about 48 h, 5 minutesto about 24 h, from about 5 minutes to about 12 hours, from about 5minutes to about 6 hours, from about 5 minutes to about 3 hours, 5minutes to 2 hours, or form about 5 minutes to about 1 hour. Inexemplary embodiments, the reaction is carried out for a period of timefrom about 5 minutes to about 1 h. In some embodiments, the reaction iscarried out until one of the conjugation partners is at least about 70%,80%, 90%, 95%, 97%, 99%, or 100% consumed.

In certain embodiments, the reaction is carried out under substantiallyoxygen free conditions.

In some embodiments, the method comprises

-   -   reacting the lipid-containing conjugation partner and an amino        acid-comprising conjugation partner to provide an amino acid or        peptide conjugate;    -   synthesising the amino acid sequence of a peptide by solid phase        peptide synthesis (SPPS);    -   coupling the amino acid of the amino acid conjugate or an amino        acid of the peptide conjugate to the solid phase bound peptide        by SPPS so as to provide a peptide conjugate comprising a        peptide epitope, a peptide conjugate comprising a linker group        or one or more amino acids thereof, or a peptide conjugate        comprising a peptide epitope bound to the amino acid to which        lipid-containing conjugation partner is conjugated via a linker        group.

In some embodiments, the method further comprises acylating the No-aminogroup of the amino acid of the amino acid conjugate or the amino acid towhich the lipid-containing conjugation partner is conjugated of any oneof the peptide conjugates.

In some embodiments, the method comprises cleaving the peptide conjugatefrom the solid phase support.

In some embodiments, the method comprises

-   -   synthesising the amino acid sequence of the peptide of the        peptide-containing conjugation partner by solid phase peptide        synthesis (SPPS); and    -   reacting the lipid-containing conjugation partner and        peptide-containing conjugation partner in accordance with any of        the embodiments described herein.

In exemplary embodiments, the method comprises synthesising the aminoacid sequence of the peptide of the peptide-containing conjugationpartner by SPPS,

-   -   cleaving the peptide from the solid phase support; and    -   reacting the lipid-containing conjugation partner and        peptide-containing conjugation partner in accordance with any of        the embodiments described herein.

In one embodiment, the peptide-containing conjugation partner is notpurified prior to reaction with the lipid-containing conjugationpartner.

In some embodiments, one or more protecting groups are removed oncleaving the peptide from the solid phase support. In certainembodiments, all of the protecting groups present in the peptide areremoved.

In one embodiment, the SPPS is Fmoc-SPPS.

In some embodiments, the amino acid residue in the peptide of thepeptide-containing conjugation partner bearing the carbon-carbon doublebond or thiol to be reacted is an N-terminal amino acid residue and themethod comprises acylating the N-terminal amino group prior to cleavingthe peptide from the solid phase. In exemplary embodiments, the aminoacid residue is an N-terminal residue. In specifically contemplatedembodiments, the N-terminal residue is a cysteine residue.

In one embodiment, the method further comprises separating the peptideconjugate from the reaction medium and optionally purifying the peptideconjugate.

In another aspect, the present invention provides a compound of theformula (V):

wherein

-   -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   R3, R4, R5, R8, and R9 are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R9 is an amino protecting        group, L3-C(O), or A2;    -   R6 and R7 at each instance of n are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl,    -   L1 is C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-6alkyl or C3-6cycloalkyl;    -   A1 and A2 are each independently an amino acid or a peptide; or        A1 is OH or OP1, wherein P1 is a carboxyl protecting group, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101; and    -   wherein any alkyl, cycloalkyl or heteroalkyl present in any of        R1, R2, R3, R4, R5, R6, R7, R8, R9, L1, and L3 is optionally        substituted, and or a pharmaceutically acceptable salt or        solvate thereof.

In another aspect, the present invention provides a compound of theformula (V):

wherein

-   -   m is an integer from 0 to 4;    -   n is 1 or 2;    -   R1 and R2 at each instance of m are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   R3, R4, R5, R8, and R9 are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl; or R9 is L3-C(O) or A2;    -   R6 and R7 at each instance of n are each independently hydrogen,        C1-6alkyl, or C3-6cycloalkyl;    -   L1 is C5-21alkyl or C4-20heteroalkyl;    -   L3 is C1-6alkyl or C3-6cycloalkyl;    -   A1 and A2 are each independently a peptide; or A1 is OH, and        wherein A1 or A2 comprise one or more EBV LMP2 epitopes, or        wherein A1, A2 or both A1 and A2 comprise one or more peptides        selected from the group consisting of SEQ ID NOs: 1-101;    -   provided that:        -   when R9 is not A2, A1 is a peptide; and        -   wherein any alkyl, cycloalkyl or heteroalkyl present in any            of R1, R2, R3, R4, R5, R6, R7, R8, R9, L1, and L3 is            optionally substituted, and or a pharmaceutically acceptable            salt or solvate thereof.

In one embodiment, m, n, R6, R7, A1 and A2 are each independently asdefined in any of the embodiments relating to the compound of formula(I).

In one embodiment, L1 is C5-21alkyl. In one embodiment, L1 isC5-21alkyl. In another embodiment, L1 is C9-21alkyl. In yet anotherembodiment, L1 is C11-21alkyl. In one exemplary embodiment, L1 is C11,C13, C15, C17, or C19alkyl. In one specifically contemplated embodiment,L1 is C15alkyl.

In one embodiment, L1 comprises a linear chain of 9-21 carbon atoms. Inone specifically contemplated embodiment, L1 is linear C15alkyl.

In one embodiment, m is an integer from 0 to 2. In another embodiment, mis 0 or 1.

In one specifically contemplated embodiment, m is 0.

In one specifically contemplated embodiment, R1 and R2 at each instanceof m are each independently hydrogen.

In one specifically contemplated embodiment, R3 is hydrogen.

In one specifically contemplated embodiment, R4 and R5 are eachhydrogen.

In one specifically contemplated embodiment, n is 1.

In one specifically contemplated embodiment, R6 and R7 are eachhydrogen.

In exemplary embodiments, R8 is hydrogen.

In some embodiments, R8 is hydrogen and R9 is hydrogen, an aminoprotecting group, L3-C(O), or A2. In one embodiment, R8 and R9 are eachhydrogen; or R9 is L3-C(O) or A2. In one exemplary embodiment R8 ishydrogen and R9 is L3-C(O). In one specifically contemplated embodiment,L3 is methyl.

In some embodiments, A1 is OP1 or OH and R9 is hydrogen, an aminoprotecting group or L3-C(O).

In some embodiments, A1 and/or A2 is an amino acid or a peptide. In someembodiments, the peptide comprises an epitope.

In some embodiments, A1 is serine or a peptide comprising serine as thefirst N-terminal amino acid residue.

In some embodiments, A1 and/or A2 is a peptide comprising a solubilisinggroup comprising an amino acid sequence comprising two or morehydrophilic amino acid residues in the peptide chain.

In some embodiments, A1 is a peptide comprising serine as the firstN-terminal amino acid residue and a solubilising group comprising anamino acid sequence comprising two or more hydrophilic amino acidresidues in the peptide chain adjacent to the serine.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; and R4 andR5 are each hydrogen.

In one embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 ishydrogen, L3-C(O), or A2. In one embodiment, n is 1; R6, R7, and R8 areeach hydrogen; and R9 is hydrogen or L3-C(O). In one embodiment, n is 1;R6, R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O), whereinL3 is methyl.

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen, L3-C(O), or A2. In one embodiment, L1 is C11-21alkyl; m is 0;R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 areeach hydrogen; R9 is hydrogen or L3-C(O).

In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 ishydrogen or L3-C(O), wherein L3 is methyl.

In some embodiments, L1 is C15 linear alkyl; m is 0; n is 1; R3, R4, R5,R6, R7, and R8 are each hydrogen; R9 is Fmoc, and A1 is OH in thecompound of the formula (V).

In some embodiments, the amino protecting group of R9 is not Fmoc. Insome embodiments, the amino protecting group of R9 is Boc.

In some embodiments, the compound of formula (V) comprises 3 or more, 4or more, or 5 or more contiguous amino acids.

In some embodiments, the amino and/or carboxyl protecting groups are asdefined in any of the embodiments relating to the compound of formula(I).

Those skilled in the art will appreciate that compound of formula (V) isa peptide conjugate and certain embodiments relating to the peptideconjugates of the conjugation method described herein also apply to thecompounds of formula (V).

In some embodiments, the compound of formula (V) is a self adjuvantingpeptide.

In some embodiments, the compound comprises a linker or one or moreamino acids thereof. In some embodiments, the peptide comprises a linkeror one or more amino acids thereof. In some embodiments, the peptidecomprises a peptide epitope bound to via a linker to the amino acid towhich L1 is bound. In some embodiments, the peptide comprises two ormore epitopes. In some embodiments, the linker is an amino acid sequencefrom about 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2 to8 amino acids in length.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an effective amount of a peptide conjugate of thepresent invention or a pharmaceutically acceptable salt or solvatethereof, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is an immunogeniccomposition.

In one embodiment, the composition does not include an extrinsicadjuvant.

In some embodiments, the composition is a vaccine.

In one embodiment, the pharmaceutical composition comprises an effectiveamount of two or more peptide conjugates of the present invention, forexample the pharmaceutical composition comprises an effective amount ofthree or more peptide conjugates of the present invention. In oneexample, the pharmaceutical composition comprises an effective amount oftwo or more peptide conjugates of the invention, wherein the two or morepeptide conjugates comprise substantially all of the immunogenic regionsof LMP2.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an effective amount of a peptide of the presentinvention or a pharmaceutically acceptable salt or solvate thereof, anda pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprises an effectiveamount of two or more peptides of the present invention, for example thepharmaceutical composition comprises an effective amount of three ormore peptides of the present invention.

In one embodiment, the pharmaceutical composition comprises an effectiveamount of one or more peptide conjugates of the present inventiontogether with one or more peptides of the present invention, or anycombination thereof. For example, the pharmaceutical compositioncomprises an effective amount of two or more peptide conjugates of thepresent invention and one or more peptides of the present invention, oran effective amount of one or more peptide conjugates of the presentinvention and two or more peptides of the present invention.

In another aspect, the present invention provides a method ofvaccinating or eliciting an immune response in a subject comprisingadministering to the subject an effective amount of a peptide conjugateor peptide of the present invention.

In another aspect, the present invention provides use of a peptideconjugate or peptide of the invention for vaccinating or eliciting animmune response in a subject.

In another aspect, the present invention provides use of a peptideconjugate or a peptide of the invention in the manufacture of amedicament for vaccinating or eliciting an immune response in a subject.

In another aspect, the present invention provides a method ofvaccinating or eliciting an immune response in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of the present invention.

In another aspect, the present invention provides use of apharmaceutical composition of the invention for vaccinating or elicitingan immune response in a subject.

In another aspect, the present invention provides use of one or morepeptides of the present invention or one or more peptide conjugates ofthe present invention in the manufacture of a medicament for vaccinatingor eliciting an immune response in a subject.

In another aspect, the present invention provides a method of elicitingan immune response in a subject comprising administering to the subjectan effective amount of a peptide conjugate of the present invention or apharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention provides use of a peptideconjugate of the invention or a pharmaceutically acceptable salt orsolvate thereof for eliciting an immune response in a subject.

In another aspect, the present invention provides use of a peptideconjugate of the invention or a pharmaceutically acceptable salt orsolvate thereof in the manufacture of a medicament for eliciting animmune response in a subject.

In another aspect, the present invention provides a method ofvaccinating a subject comprising administering to the subject aneffective amount of a peptide conjugate of the present invention or apharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention provides use of a peptideconjugate of the present invention for vaccinating a subject or apharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention provides use of a peptideconjugate of the invention or a pharmaceutically acceptable salt orsolvate thereof in the manufacture of a medicament for vaccinating asubject.

In some embodiments, the method comprises the administration of one ormore peptides of the present invention and/or one or more peptideconjugates of the present invention, for example one or more peptides incombination with one or more peptide conjugates to the subject.

In some embodiments, one or more peptides of the present inventionand/or one or more peptide conjugates of the present invention, forexample one or more peptides in combination with one or more peptideconjugates are used for vaccinating or eliciting an immune response inthe subject or in the manufacture of a medicament for vaccinating oreliciting an immune response in the subject.

In some embodiment, two or more peptides, two or more peptideconjugates, or one or more peptides and one or more peptide conjugatesare used or administered. In some embodiments the two or more peptides,two or more peptide conjugates, or one or more peptides and one or morepeptide conjugates are used or administered simultaneously,sequentially, or separately.

Asymmetric centers may exist in the compounds described herein. Theasymmetric centers may be designated as (R) or (S), depending on theconfiguration of substituents in three dimensional space at the chiralcarbon atom. All stereochemical isomeric forms of the compounds,including diastereomeric, enantiomeric, and epimeric forms, as well asd-isomers and l-isomers, and mixtures thereof, includingenantiomerically enriched and diastereomerically enriched mixtures ofstereochemical isomers, are within the scope of the invention.

Individual enantiomers can be prepared synthetically from commerciallyavailable enantiopure starting materials or by preparing enantiomericmixtures and resolving the mixture into individual enantiomers.Resolution methods include conversion of the enantiomeric mixture into amixture of diastereomers and separation of the diastereomers by, forexample, recrystallization or chromatography, and any other appropriatemethods known in the art. Starting materials of defined stereochemistrymay be commercially available or made and, if necessary, resolved bytechniques well known in the art.

The compounds described herein may also exist as conformational orgeometric isomers, including cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers. All such isomers and any mixtures thereof arewithin the scope of the invention.

Also within the scope of the invention are any tautomeric isomers ormixtures thereof of the compounds described. As would be appreciated bythose skilled in the art, a wide variety of functional groups and otherstructures may exhibit tautomerism. Examples include, but are notlimited to, keto/enol, imine/enamine, and thioketone/enethioltautomerism.

The compounds described herein may also exist as isotopologues andisotopomers, wherein one or more atoms in the compounds are replacedwith different isotopes.

Suitable isotopes include, for example, ¹H, ²H (D), ³H (T), ¹²C, ¹³C,¹⁴C, ¹⁶O, and ¹⁸O. Procedures for incorporating such isotopes into thecompounds described herein will be apparent to those skilled in the art.Isotopologues and isotopomers of the compounds described herein are alsowithin the scope of the invention.

Also within the scope of the invention are pharmaceutically acceptablesalts and solvates, including hydrates of the compounds describedherein. Such salts include, acid addition salts, base addition salts,and quaternary salts of basic nitrogen-containing groups.

Acid addition salts can be prepared by reacting compounds, in free baseform, with inorganic or organic acids. Examples of inorganic acidsinclude, but are not limited to, hydrochloric, hydrobromic, nitric,sulfuric, and phosphoric acid. Examples of organic acids include, butare not limited to, acetic, trifluoroacetic, propionic, succinic,glycolic, lactic, malic, tartaric, citric, ascorbic, maleic, fumaric,pyruvic, aspartic, glutamic, stearic, salicylic, methanesulfonic,benzenesulfonic, isethionic, sulfanilic, adipic, butyric, and pivalic.

Base addition salts can be prepared by reacting compounds, in free acidform, with inorganic or organic bases. Examples of inorganic baseaddition salts include alkali metal salts, alkaline earth metal salts,and other physiologically acceptable metal salts, for example,aluminium, calcium, lithium, magnesium, potassium, sodium, or zincsalts. Examples of organic base addition salts include amine salts, forexample, salts of trimethylamine, diethylamine, ethanolamine,diethanolamine, and ethylenediamine.

Quaternary salts of basic nitrogen-containing groups in the compoundsmay be may be prepared by, for example, reacting the compounds withalkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides, and iodides, dialkyl sulfates such as dimethyl, diethyl,dibutyl, and diamyl sulfates, and the like.

The general chemical terms used in the formulae herein have their usualmeaning.

The term “aliphatic” is intended to include saturated and unsaturated,nonaromatic, straight chain, branched, acyclic, and cyclic hydrocarbons.Those skilled in the art will appreciate that aliphatic groups include,for example, alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenylgroups. In some embodiments, the aliphatic group is saturated.

The term “heteroaliphatic” is intended to include aliphatic groups,wherein one or more chain carbon atoms are replaced with a heteroatom.In some embodiments, the heteroaliphatic is saturated.

The term “alkyl” is intended to include saturated or unsaturatedstraight chain and branched chain hydrocarbon groups. Examples ofsaturated hydrocarbon groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, and the like. Unsaturated alkylgroups have one or more carbon-carbon double bonds or triple bonds.Examples of unsaturated alkyl groups include vinyl, prop-2-enyl, crotyl,isopent-2-enyl, 2-butadienyl, penta-2,4-dienyl, penta-1,4-dienyl,ethynyl, prop-3-ynyl, but-3-ynyl, and the like. In some embodiments, thealkyl is saturated.

The term “heteroalkyl” is intended to include alkyl groups, wherein oneor more chain carbon atoms are replaced with a heteroatom. In someembodiments, the heteroalkyl is saturated.

The term “cycloalkyl” is intended to include non-aromatic cyclic alkylgroups. Examples of cycloalkyl groups include but are not limited tocyclopentyl, cyclohexyl, cyclohex-1-enyl, cyclohex-3-enyl, cycloheptyl.In some embodiments, the cycloalkyl is saturated.

The term “heteroatom” is intended to include oxygen, nitrogen, sulfur,or phosphorus. In some embodiments, the heteroatom is selected from thegroup consisting of oxygen, nitrogen, and sulfur.

The term “aryl” is intended to include aromatic radicals. Examplesinclude, but are not limited to, phenyl, tolyl, naphthyl, indanyl, andthe like. In some embodiments, aryl groups comprise from 4 to 8 or from6 to 8 carbon atoms in the aromatic ring system.

As used herein, the term “substituted” is intended to mean that one ormore hydrogen atoms in the group indicated is replaced with one or moreindependently selected suitable substituents, provided that the normalvalency of each atom to which the substituent/s are attached is notexceeded, and that the substitution results in a stable compound.

Examples of optional substituents for aliphatic, heteroaliphatic, alkyl,heteroalkyl, and cycloalkyl groups in the compounds described hereininclude but are not limited to halo, CN, NO₂, OH, NH₂, NHR1, NR1R2,C1-6haloalkyl, C1-6haloalkoxy, C(O)NH₂, C(O)NHR1, C(O)NR1R1, SO₂R1, OR1,SR1, S(O)R1, C(O)R1, and C1-6aliphatic; wherein R1 and R2 are eachindependently C1-6alkyl.

The term “carboxyl protecting group” as used herein is means a groupthat is capable of readily removed to provide the OH group of a carboxylgroup and protects the carboxyl group against undesirable reactionduring synthetic procedures. Such protecting groups are described inProtective Groups in Organic Synthesis edited by T. W. Greene et al.(John Wiley & Sons, 1999) and ‘Amino Acid-Protecting Groups’ by FernandoAlbericio (with Albert Isidro-Llobet and Mercedes Alvarez) ChemicalReviews 2009 (109) 2455-2504. Examples include, but are not limited to,alkyl and silyl groups, for example methyl, ethyl, tert-butyl,methoxymethyl, 2,2,2-trichloroethyl, benzyl, diphenylmethyl,trimethylsilyl, and tert-butyldimethylsilyl, and the like.

The term “amine protecting group” as used herein means a group that iscapable of being readily removed to provide the NH₂ group of an aminegroup and protects the amine group against undesirable reaction duringsynthetic procedures. Such protecting groups are described in ProtectiveGroups in Organic Synthesis edited by T. W. Greene et al. (John Wiley &Sons, 1999) and ‘Amino Acid-Protecting Groups’ by Fernando Albericio(with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009(109) 2455-2504. Examples include, but are not limited to, acyl andacyloxy groups, for example acetyl, chloroacetyl, trichloroacetyl,o-nitrophenylacetyl, o-nitrophenoxy-acetyl, trifluoroacetyl,acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinoyl, aminocaproyl,benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl,2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl,tert-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2,4-dichloro-benzyloxycarbonyl, and the like. Further examples includeCbz (carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl), Bpoc(2-(4-biphenyl)isopropoxycarbonyl) and Dde(1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl).

As used herein, the term “and/or” means “and”, or “or”, or both.

The term “(s)” following a noun contemplates the singular and pluralform, or both.

The term “comprising” as used in this specification means “consisting atleast in part of”. When interpreting each statement in thisspecification that includes the term “comprising”, features other thanthat or those prefaced by the term may also be present. Related termssuch as “comprise” and “comprises” are to be interpreted in the samemanner. The “containing” is also to be interpreted in the same manner.

The invention may also be said broadly to consist in the parts, elementsand features referred to or indicated in the specification of theapplication, individually or collectively, in any or all combinations oftwo or more of said parts, elements or features, and where specificintegers are mentioned herein which have known equivalents in the art towhich the invention relates, such known equivalents are deemed to beincorporated herein as if individually set forth.

It is intended that reference to a range of numbers disclosed herein(for example, 1 to 10) also incorporates reference to all rationalnumbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,7, 8, 9, and 10) and also any range of rational numbers within thatrange (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore,all sub-ranges of all ranges expressly disclosed herein are herebyexpressly disclosed. These are only examples of what is specificallyintended and all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

Although the present invention is broadly as defined above, thosepersons skilled in the art will appreciate that the invention is notlimited thereto and that the invention also includes embodiments ofwhich the following description gives examples.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described with reference to the accompanyingfigures in which:

FIG. 1 shows an RP-HPLC trace of LMP2 S1, as described herein in Example4.

FIG. 2 shows an RP-HPLC trace of LMP2 S2, as described herein in Example4.

FIG. 3 shows an RP-HPLC trace of Pam1-C(Ac)SK4-LMP2 S4, as describedherein in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to amino acid and peptide conjugates, andmethods of making peptide conjugates and using the peptides and peptideconjugates in immunotherapeutic treatments, particularlyimmunotherapeutic treatments of conditions associated with EBV.Particular methods of making the conjugates comprises reacting anlipid-containing conjugation partner and an amino acid-comprisingconjugation partner under conditions effective to conjugate thelipid-containing conjugation partner to the amino acid-comprisingconjugation partner in a thiol-ene reaction. In some embodiments, themethod comprises reacting an lipid-containing conjugation partner and apeptide-containing conjugation partner under conditions effective toconjugate the lipid-containing conjugation partner to the peptide of thepeptide-containing conjugation partner in a thiol-ene reaction.

The thiol-ene reaction involves the addition of a thiol across anon-aromatic carbon-carbon double bond (i.e. hydrothiolation of thecarbon-carbon double bond). The reaction proceeds via a free radicalmechanism. There are three distinct phases in the reaction: initiation,polymerisation or coupling, and termination. Radical generation givesrise to an electrophilic thiyl radical which propagates across the enegroup, forming a carbon-centred radical. Chain transfer from anadditional thiol molecule then quenches the radical on carbon to givethe final product.

In the method the present invention, one conjugation partner comprisesthe thiol and the other comprises the carbon carbon double bond.

One or more free radicals may be generated in the method of the presentinvention by any method known in the art. The free radicals may begenerated thermally and/or photochemically. One or more free radicalinitiators may be used to initiate the generation of free radicals.Suitable free radical initiators include thermal initiators andphotoinitiators.

Free radicals are generated from thermal initiators by heating. The rateof degradation of the thermal initiator and resulting free radicalformation depends on the initiator and the temperature at which theinitiator is heated. Higher temperatures generally result in fasterdecomposition. A person skilled in the art will be able to select anappropriate temperature for heating the initiator without undueexperimentation.

Numerous thermal initiators are commercially available. Examples ofthermal initiators include but are not limited to tert-amylperoxybenzoate, 1,1′-azobis(cyclohexanecarbonitrile),2,2′-azobisisobutyronitrile (AIBN), benzoyl peroxide, tert-butylhydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butylperoxybenzoate, tert-butylperoxy isopropyl carbonate, lauroyl peroxide,peracetic acid, and potassium persulfate.

Free radicals may be generated from photoinitiators by irradiation withlight. The frequency of light necessary to induce degradation of thephotoinitiators and free radical formation depends on the initiator.Many photoinitiators can be initiated with ultraviolet light.

Light of a specific wavelength or wavelength range may be used toselectively irradiate the initiator, where the lipid-containingconjugation partner or amino acid-comprising conjugation partner, forexample a peptide-containing conjugation partner, comprisesphotosensitive groups. In certain embodiments of the method of thepresent invention, a frequency of about 365 nm is used. Light of thisfrequency is generally compatible with the side chains of naturallyoccurring amino acids.

A wide range of photoinitiators are commercially available. Examples ofphotoinitiators include but are not limited to acetophenone, anisoin,anthraquinone, anthraquinone-2-sulfonic acid, benzil, benzoin, benzoinethyl ether, benzoin isobutyl ether, benzoin methyl ether, benzophenone,3,3′,4,4′-benzophenonetetracarboxylic dianhydride, 4-benzoylbiphenyl,2-benzyl-2-(dimethylamino)-4′-morpholinobutyrophenone,4′-bis(diethylamino)benzophenone, 4,4′-bis(dimethylamino)benzophenone,camphorquinone, 2-chlorothioxanthen-9-one, dibenzosuberenone,2,2-diethoxyacetophenone, 4,4′-dihydroxybenzophenone,2,2-dimethoxy-2-phenylacetophenone (DMPA),4-(dimethylamino)benzophenone, 4,4′-dimethylbenzil,2,5-dimethylbenzophenone, 3,4-dimethylbenzophenone,4′-ethoxyacetophenone, 2-ethylanthraquinone, 3′-hydroxyacetophenone,4′-hydroxyacetophenone, 3-hydroxybenzophenone, 4-hydroxybenzophenone,1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methylpropiophenone,2-methylbenzophenone, 3-methylbenzophenone, methybenzoylformate,2-methyl-4′-(methylthio)-2-morpholinopropiophenone, phenanthrenequinone,4′-phenoxyacetophenone, and thioxanthen-9-one.

A person skilled in the art will be able to select appropriate freeradical initiators for use in the method having regard to, for example,the nature of the lipid-containing conjugation partner, aminoacid-comprising conjugation partner, for example a peptide-containingconjugation partner, and any other components present in the reactionmixture. In some embodiments, the initiator is present in the reactionin a stoichiometric ratio relative to the starting material comprisingthe thiol of from about 20:1 to about 0.05:1, from about 10:1 to about0.05:1, from about 5:1 to about 0.05:1, from about 3:1 to about 0.5:1.

The lipid-containing conjugation partner and amino acid-comprisingconjugation partner, for example a peptide-containing conjugationpartner, in the reaction are as defined in any of the embodimentsdescribed herein.

The lipid-containing conjugation partner and amino acid-comprisingconjugation partner, for example a peptide-containing conjugationpartner, may be prepared using known synthetic chemistry techniques (forexample, the methods generally described in Louis F Fieser and Mary F,Reagents for Organic Synthesis v. 1-19, Wiley, New York (1967-1999 ed.)or Beilsteins Handbuch der organischen Chemie, 4, Aufl. Ed.Springer-Verlag Berlin, including supplements (also available via theBeilstein online database)) or, in some embodiments, may be commerciallyavailable.

Lipid-Containing Conjugation Partner Compounds of the Formula (II)

wherein m, R1, R2, R3, R4, R5, and L1 are each independently as definedin any of the embodiments described for the compound of formula (I) maybe prepared by reacting a compound of the formula (VI)L¹-C(O)—X   (VI)

wherein X is OH or a suitable leaving group with a compound of theformula (VII):

under conditions effective for esterification. Methods foresterification are well known in the art. For example, when X is chloro,the reaction may be carried out in the presence of a base, such aspyridine or triethylamine, in a suitable solvent. The acid chloride maybe converted in situ to a more reactive species (e.g. to thecorresponding iodide, using sodium iodide). The temperature at which thereaction is carried out depends on the reactivity of the acid speciesand the solvent used.

Numerous compounds of formula (VI) are commercially available. Othersmay be prepared using standard synthetic chemistry techniques fromcommercially available precursors. For example, compounds of formula(VI) wherein X is chloro may be prepared treating the correspondingcarboxylic acid with thionyl chloride in a suitable solvent or mixtureof solvents.

Lipid Containing Conjugation Partner Compounds of the Formula (IIA)

wherein p, R11, R22, R33, R44, and L1 are as defined in the compound offormula (IA) may be prepared by reacting a compound of the formula (VI)as defined above with a compound of the formula (VIII):

wherein P is a suitable protecting group under conditions effective foresterification, and then removing the protecting group.

Alternatively, compounds of the formula (IIA) may be prepared byreacting a compound of the formula (VI) as defined above with a compoundof the formula (IX):

wherein P is a suitable protecting group under conditions effective foresterification, removing the protecting group, and then converting thecorresponding alcohol to a thiol. Suitable methods for converting thealcohol to a thiol will be apparent to those skilled in the art.

Preparation of the compounds may involve the protection and deprotectionof various chemical groups. The need for protection and deprotection,and the selection of appropriate protecting groups, can be readilydetermined by a person skilled in the art.

Protecting groups and methods for protection and deprotection are wellknown in the art (see e.g. T. W. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., Wiley & Sons, Inc., New York(1999)).

Similarly, compounds of formula (VII), (VIII), and (IX) are alsocommercially available or may be prepared from commercially availableprecursors using standard synthetic chemistry techniques.

The order in which the lipid-containing conjugation partner and aminoacid-comprising conjugation partner, for example a peptide-containingconjugation partner, and any other components present in the reactionmixture are introduced into the reaction vessel may vary. The reactionmay be carried out as a one-pot procedure.

The stoichiometry of the lipid-containing conjugation partner and aminoacid-comprising conjugation partner, for example a peptide-containingconjugation partner, in the reaction may vary. In some embodiments, thestoichiometric ratio of amino acid-comprising conjugation partner tolipid-containing conjugation partner is from about 1:0.5 to about 1:20,from about 1:1 to about 1:10, from about 1:1 to about 1:5, from about1:1 to about 1:3. In some embodiments, the stoichiometric ratio ofpeptide-containing conjugation partner to lipid-containing conjugationpartner is from about 1:0.5 to about 1:20, from about 1:1 to about 1:10,from about 1:1 to about 1:5, from about 1:1 to about 1:3.

The reaction may be carried out at any suitable temperature. In someembodiments, the reaction is carried out at a temperature from about−25° C. to about 200° C., from about −10° C. to about 150° C., fromabout 0° C. to about 125° C., from about ambient temperature to about100° C. In some embodiments, the reaction is carried out at atemperature of less than about 200° C., less than about 175° C., lessthan about 150° C., less than about 125° C., or less than about 100° C.

In some embodiments, the reaction is carried out at a temperature aboveambient temperature. In one embodiment, the reaction is carried out at atemperature from 40 to 200° C., from 50 to 150° C., from 60 to 100° C.,from 65 to 90° C., or from 70 to 80° C. In some embodiments, thereaction is carried out at a temperature greater than 40° C., greaterthan 50° C., greater than 75° C., greater than 100° C., or greater than150° C.

The temperature at which the reaction is carried out may depend on howfree radicals are generated in the reaction. The temperature used may beselected to control the rate of the reaction. The temperature may beadjusted during the course of the reaction to control the rate of thereaction. By controlling the rate of the reaction it may be possible tominimise or obviate the formation of undesirable by products (e.g.telomerisation or polymerisation products).

If free radicals are generated thermally (e.g. using a thermalinitiator), the reaction will generally be carried out at a temperatureabove ambient temperature. The temperature will depend on the reactivityof the species from which free radicals are generated.

If free radicals are generated photochemically the reaction may becarried out, advantageously, at ambient temperature. In certainembodiments, it may be desirable to cool the reaction mixture to slowthe rate of reaction or conversely heat the reaction mixture to increasethe rate of reaction.

A person skilled in the art will be able to select appropriatetemperatures for carrying out the method having regard to the reactivityof the lipid-containing conjugation partner, amino acid-comprisingconjugation partner, for example a peptide-containing conjugationpartner, and free radical initiator if used.

The temperature at which the reaction is carried out may be controlledby heating or cooling the reaction mixture. The temperature of thereaction mixture may be controlled by suitable method known in the art.Heat may be applied to the reaction mixture, for example, using a heatexchanger within the reaction vessel, a heating jacket surrounding thereaction vessel, or by immersing the reaction vessel in a heated liquid(e.g. an oil or sand bath). In certain exemplary embodiments, thereaction mixture is heated by microwave irradiation.

The progress of the reaction may be monitored by any suitable means, forexample, by thin layer chromatography (TLC) or high performance liquidchromatography (HPLC). The reaction may be allowed to proceed tosubstantial completion, as monitored by the consumption of at least oneof the starting materials. In some embodiments, the reaction is allowedto proceed for a period of time from 1 minute to 7 days, 5 minutes to 72hours, 10 minutes to 48 hours, 10 minutes to 24 hours. In otherembodiments, the reaction is allowed to proceed for a period of timeless than 72 h, less than 48 h, less than 24 h, less than 12 h, lessthan 6 h, less than 4 h, less than 2 h, or less than 1 h.

In some embodiments, the reaction is carried out until at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 99% of the lipid-containingconjugation partner or amino acid-comprising conjugation partner,whichever is stoichiometrically less, has been consumed. In someembodiments, the reaction is carried out until at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 97%, at least about 99% of the lipid-containing conjugationpartner or peptide-containing conjugation partner, whichever isstoichiometrically less, has been consumed. The consumption of startingmaterials may be monitored by any suitable method, for example, HPLC.

The reaction mixture may be mixed by any suitable method known in theart, for example, using a magnetic or mechanical stirrer. The methodused may depend on the scale on which the reaction is carried out.

The reaction is generally carried out in a liquid reaction medium. Theliquid reaction medium may comprise a solvent. Examples of suitablesolvents include dimethylformamide, dichloromethane, 1,2-dichloroethane,chloroform, carbon tetrachloride, water, methanol, ethanol,dimethylsulfoxide, trifluoroacetic acid, acetic acid, acetonitrile, andmixtures thereof.

The solvent may be selected based on the solubility of thelipid-containing conjugation partner and amino acid-comprisingconjugation partner, for example a peptide-containing conjugationpartner, in the solvent. The solubility of the free radical initiatormay also be relevant. In some embodiments, the lipid-containingconjugation partner is hydrophobic. The hydrophobicity or hydrophilicityof an amino acid-comprising conjugation partner, for example apeptide-containing conjugation partner, may vary depending on, forexample, amino acid sequence of the peptide of a peptide-containingconjugation partner. The presence of a solubilising group in thepeptide-containing conjugation partner may increase solubility in polarsolvents, such as water. A person skilled in the art will be able toselect an appropriate solvent without undue experimentation.

The reaction may be carried out under substantially oxygen-freeconditions. Oxygen may quench free radicals formed in the reaction. Thereaction mixture may be degassed with an inert gas (e.g. nitrogen orargon) that is substantially oxygen-free to remove any dissolved oxygenbefore free radicals are generated. Alternatively, individual componentsof the reaction mixture may be degassed with inert gas that issubstantially oxygen-free prior to being combined in the reactionvessel. The reaction may be carried out under an atmosphere of inert gasthat is substantially oxygen-free.

The method of the present invention may be carried out at ambientpressure.

If the rate of chain transfer relative to propagation in the thiol-enereaction is slow, undesirable dimerisation, telomerisation, orpolymerisation may occur.

An additive that inhibits dimerisation, telomerisation, orpolymerisation may be included in the reaction mixture in the method ofthe present invention. The inventors have found that in some embodimentsthe inclusion of an extraneous thiol that facilitates chain transfer asan additive in the reaction mixture reduces the formation of undesirableby products. The extraneous thiol may, in some embodiments, increase theefficiency of the desired thiol ene reaction. Examples of suitableextraneous thiols include but are not limited to reduced glutathione,DODT, DTT, protein, and the like. The inventors have found that in someembodiments the inclusion of DTT resulted in no undesirable by products.

In certain embodiments, the extraneous thiol is a sterically hinderedthiol. Non-limiting examples of a suitable sterically hinderedextraneous thiol include tert-butyl mercaptan and 1-methylpropylmercaptan.

The inclusion of an acid in some embodiments may also inhibitdimerisation, telomerisation, or polymerisation. The acid may be astrong inorganic acid, for example HCl, or organic acid, for exampleTFA. In certain embodiments, the additive is TFA.

The inventors have found that in some embodiments including bothtert-butyl mercaptan and TFA as additives in the reaction mixture canreduce the the formation of oligomers, and increase the conversion ofstarting material to the desired product. Accordingly, in certainexemplary embodiments, the reaction mixture comprises a combination ofTFA and tert-butyl mercaptan.

The additive is generally used in an amount sufficient to minimise theformation of undesirable by products without adversely affecting thereaction or any, optional, subsequent steps in the method. In someembodiments, the additive is present in the reaction a stoichiometricratio relative to the starting material comprising the thiol of fromabout 20:1 to about 0.05:1, from about 10:1 to about 0.5:1, from about5:1 to about 1:1, from about 3:1 to about 1:1.

In some embodiments, less than about 50%, less than about 40%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, less than about 3%, orless than about 1% by weight of the lipid-containing conjugation partnerand amino acid-comprising conjugation partner starting materials used inthe reaction are undesirable by products resulting from dimerisation,telomerisation, or polymerisation. In some embodiments, less than about50%, less than about 40%, less than about 30%, less than about 25%, lessthan about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 3%, or less than about 1% by weight of thelipid-containing conjugation partner and peptide-containing conjugationpartner starting materials used in the reaction are undesirable byproducts resulting from dimerisation, telomerisation, or polymerisation.The purity of the products of the reaction may be determined by, forexample, HPLC.

The concentration of the lipid-containing conjugation partner and aminoacid-comprising conjugation partner, for example a peptide-containingconjugation partner, respectively, in the reaction mixture may alsoaffect the reaction. Those skilled in the art will be able to vary theconcentration of the lipid-containing conjugation partner andpeptide-containing conjugation partner in the reaction mixture to e.g.optimise yield and purity without undue experimentation.

In some embodiments, the starting material comprising the thiol ispresent in a concentration from about 0.05 mM to about 1 M, from about0.5 mM to about 1 M, from about 1 mM to about 1 M. In some embodiments,the concentration is at least about 0.05 mM, 0.5 mM, or 1 mM.

In some embodiments, the concentration of the starting materialcomprising the alkene is at least about 0.05 mM, 0.5 mM, or 1 mM.

In some embodiments, the amino acid conjugate or peptide conjugate maybe separated from the reaction medium after the reaction and optionallypurified. In some embodiments, the peptide conjugate may be separatedfrom the reaction medium after the reaction and optionally purified. Theconjugate may be separated from the reaction medium using any suitablemethod known in the art, for example, by precipitation.

In some embodiments, the amino acid or peptide conjugate is purifiedafter separating it from the reaction medium. In some embodiments, thepeptide conjugate is purified after separating it from the reactionmedium. In specifically contemplated embodiments, the conjugate ispurified by HPLC using one or more suitable solvents.

The peptide conjugate produced by and/or the peptide-containingconjugation partner in the method of the present invention may comprisea synthetic peptide. Synthetic peptides may be prepared using solidphase peptide synthesis (SPPS).

The basic principle for solid phase peptide synthesis (SPPS) is astepwise addition of amino acids to a growing polypeptide chain anchoredvia a linker molecule to a solid phase support, typically a resinparticle, which allows for cleavage and purification once thepolypeptide chain is complete. Briefly, a solid phase resin support anda starting amino acid are attached to one another via a linker molecule.Such resin-linker-acid matrices are commercially available.

The amino acid to be coupled to the resin is protected at itsNo-terminus by a chemical protecting group.

The amino acid may also have a side-chain protecting group. Suchprotecting groups prevent undesired or deleterious reactions from takingplace during the process of forming the new peptide bond between thecarboxyl group of the amino acid to be coupled and the unprotectedNo-amino group of the peptide chain attached to the resin.

The amino acid to be coupled is reacted with the unprotected No-aminogroup of the N-terminal amino acid of the peptide chain, increasing thechain length of the peptide chain by one amino acid. The carboxyl groupof the amino acid to be coupled may be activated with a suitablechemical activating agent to promote reaction with the No-amino group ofthe peptide chain. The No-protecting group of N-terminal amino acid ofthe peptide chain is then removed in preparation for coupling with thenext amino acid residue. This technique consists of many repetitivesteps making automation attractive whenever possible. Those skilled inthe art will appreciate that peptides may be coupled to the No-aminogroup of the solid phase bound amino acid or peptide instead of anindividual amino acid, for example where a convergent peptide synthesisis desired.

When the desired sequence of amino acids is achieved, the peptide iscleaved from the solid phase support at the linker molecule.

SPPS may be carried out using a continuous flow method or a batch flowmethod. Continuous flow permits real-time monitoring of reactionprogress via a spectrophotometer, but has two distinct disadvantages—thereagents in contact with the peptide on the resin are diluted, and scaleis more limited due to physical size constraints of the solid phaseresin. Batch flow occurs in a filter reaction vessel and is usefulbecause reactants are accessible and can be added manually orautomatically.

The types of protecting groups are commonly used for protecting theN-alpha-amino terminus: “Boc” (tert-butyloxycarbonyl) and “Fmoc”(9-fluorenylmethyloxycarbonyl). Reagents for the Boc method arerelatively inexpensive, but they are highly corrosive and requireexpensive equipment and more rigorous precautions to be taken. The Fmocmethod, which uses less corrosive, although more expensive, reagents istypically preferred.

For SPPS, a wide variety of solid support phases are available. Thesolid phase support used for synthesis can be a synthetic resin, asynthetic polymer film or a silicon or silicate surface (e.g. controlledpore glass) suitable for synthesis purposes. Generally, a resin is used,commonly polystyrene suspensions, or polystyrene-polyethyleneglycol, orpolymer supports for example polyamide. Examples of resinsfunctionalized with linkers suitable for Boc-chemistry include PAMresin, oxime resin SS, phenol resin, brominated Wang resin andbrominated PPOA resin. Examples of resins suitable for Fmoc chemistryinclude AMPB-BHA resin, Sieber amide resin, Rink acid resin, Tentagel SAC resin, 2-chlorotrityl chloride resin, 2-chlorotrityl alcohol resin,TentaGel S Trt-OH resin, Knorr-2-chlorotrityl resin,hydrazine-2-chlorotrityl resin, ANP resin, Fmoc photolabile resin,HMBA-MBHA resin, TentaGel S HMB resin, Aromatic Safety Catch resinBAlresin and Fmoc-hydroxylamine 2 chlorotrityl resin. Other resins includePL Cl-Trt resin, PL-Oxime resin and PL-HMBA Resin.

For each resin appropriate coupling conditions are known in theliterature for the attachment of the starting monomer or sub-unit.

Preparation of the solid phase support includes solvating the support inan appropriate solvent (e.g. dimethylformamide). The solid phasetypically increases in volume during solvation, which in turn increasesthe surface area available to carry out peptide synthesis.

A linker molecule is then attached to the support for connecting thepeptide chain to the solid phase support. Linker molecules are generallydesigned such that eventual cleavage provides either a free acid oramide at the C-terminus. Linkers are generally not resin-specific.Examples of linkers include peptide acids for example4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine (HMP), or peptideamides for example benzhydrylamine derivatives.

The first amino acid of the peptide sequence may be attached to thelinker after the linker is attached to the solid phase support orattached to the solid phase support using a linker that includes thefirst amino acid of the peptide sequence. Linkers that include aminoacids are commercially available.

The next step is to deprotect the No-amino group of the first aminoacid. For Fmoc SPPS, deprotection of the No-amino group may be carriedout with a mild base treatment (piperazine or piperidine, for example).Side-chain protecting groups may be removed by moderate acidolysis(trifluoroacetic acid (TFA), for example). For Boc SPPS, deprotection ofthe No-amino group may be carried out using for example TFA.

Following deprotection, the amino acid chain extension, or coupling,proceeds by the formation of peptide bonds. This process requiresactivation of the C-α-carboxyl group of the amino acid to be coupled.This may be accomplished using, for example, in situ reagents, preformedsymmetrical anhydrides, active esters, acid halides, orurethane-protected N-carboxyanhydrides. The in situ method allowsconcurrent activation and coupling. Coupling reagents includecarbodiimide derivatives, for example N,N′-dicyclohexylcarbodiimide orN,N-diisopropylcarbodiimide. Coupling reagents also include uronium orphosphonium salt derivatives of benzotriazol. Examples of such uroniumand phosphonium salts include HBTU(O-1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), BOP(benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate), PyBOP(Benzotriazole-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate),PyAOP, HCTU(O-(1H-6-chloro-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TCTU(O-1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), TOTU(O-[cyano(ethoxycarbonyl)methyleneamino]-N,N,N′,N″-tetramethyluroniumtetrafluoroborate), and HAPyU(O-(benzotriazol-1-yl)oxybis-(pyrrolidino)-uronium hexafluorophosphate.In some embodiments, the coupling reagent is HBTU, HATU, BOP, or PyBOP.

After the desired amino acid sequence has been synthesized, the peptideis cleaved from the resin. The conditions used in this process depend onthe sensitivity of the amino acid composition of the peptide and theside-chain protecting groups. Generally, cleavage is carried out in anenvironment containing a plurality of scavenging agents to quench thereactive carbonium ions that originate from the protective groups andlinkers. Common cleaving agents include, for example, TFA and hydrogenfluoride (HF). In some embodiments, where the peptide is bound to thesolid phase support via a linker, the peptide chain is cleaved from thesolid phase support by cleaving the peptide from the linker.

The conditions used for cleaving the peptide from the resin mayconcomitantly remove one or more side-chain protecting groups.

The use of protective groups in SPPS is well established. Examples ofcommon protective groups include but are not limited to acetamidomethyl(Acm), acetyl (Ac), adamantyloxy (AdaO), benzoyl (Bz), benzyl (Bzl),2-bromobenzyl, benzyloxy (BzlO), benzyloxycarbonyl (Z), benzyloxymethyl(Bom), 2-bromobenzyloxycarbonyl (2-Br—Z), tert-butoxy (tBuO),tert-butoxycarbonyl (Boc), tert-butoxymethyl (Bum), tert-butyl (tBu),tert-butylthio (tButhio), 2-chlorobenzyloxycarbonyl (2-Cl—Z),cyclohexyloxy (cHxO), 2,6-dichlorobenzyl (2,6-DiCl-Bzl),4,4′-dimethoxybenzhydryl (Mbh),1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene) 3-methyl-butyl (ivDde),4-{N-[1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-methylbutyl]-amino)benzyloxy (ODmab), 2,4-dinitrophenyl (Dnp), fluorenylmethoxycarbonyl(Fmoc), formyl (For), mesitylene-2-sulfonyl (Mts), 4-methoxybenzyl(MeOBzl), 4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr),4-methoxytrityl (Mmt), 4-methylbenzyl (MeBzl), 4-methyltrityl (Mtt),3-nitro-2-pyridinesulfenyl (Npys),2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),2,2,5,7,8-pentamethyl-chromane-6-sulfonyl (Pmc), tosyl (Tos),trifluoroacetyl (Tfa), trimethylacetamidomethyl (Tacm), trityl (Trt) andxanthyl (Xan).

Where one or more of the side chains of the amino acids of the peptidecontains functional groups, such as for example additional carboxylic,amino, hydroxy or thiol groups, additional protective groups may benecessary. For example, if the Fmoc strategy is used, Mtr, Pmc, Pbf maybe used for the protection of Arg; Trt, Tmob may be used for theprotection of Asn and Gin; Boc may be used for the protection of Trp andLys; tBu may be used for the protection of Asp, Glu, Ser, Thr and Tyr;and Acm, tBu, tButhio, Trt and Mmt may be used for the protection ofCys. A person skilled in the art will appreciate that there are numerousother suitable combinations.

The methods for SPPS outlined above are well known in the art. See, forexample, Atherton and Sheppard, “Solid Phase Peptide Synthesis: APractical Approach,” New York: IRL Press, 1989; Stewart and Young:“Solid-Phase Peptide Synthesis 2nd Ed.,” Rockford, Ill.: Pierce ChemicalCo., 1984; Jones, “The Chemical Synthesis of Peptides,” Oxford:Clarendon Press, 1994; Merrifield, J. Am. Soc. 85:2146-2149 (1963);Marglin, A. and Merrifield, R. B. Annu. Rev. Biochem. 39:841-66 (1970);and Merrifield R. B. JAMA. 210(7):1247-54 (1969); and “Solid PhasePeptide Synthesis—A Practical Approach” (W. C. Chan and P. D. White,eds. Oxford University Press, 2000). Equipment for automated synthesisof peptides or polypeptides is readily commercially available fromsuppliers such as Perkin Elmer/Applied Biosystems (Foster City, Calif.)and may be operated according to the manufacturer's instructions.

Following cleavage from the resin, the peptide may be separated from thereaction medium, e.g. by centrifugation or filtration. The peptide maythen be subsequently purified, e.g. by HPLC using one or more suitablesolvents.

Advantageously, the inventors have found that in some embodiments thepeptide-containing conjugation partner may be used in the method of thepresent invention without purification following cleavage of the peptidefrom the resin.

The inventors have also advantageously found that the method of thepresent invention can be carried out using a peptide-containingconjugation partner, wherein the peptide does not contain an No-aminogroup protecting group or any side chain protecting groups. The reactionis generally selective for reaction of a thiol and a non-aromaticcarbon-carbon double bond.

It may be necessary to protect thiol groups present in thepeptide-containing conjugation partner (e.g. in cysteine residues of thepeptide) with a protective group to prevent undesirable competingreactions in the method of the present invention. The thiol groups maybe protected with a protective group that is not removable under theconditions used to remove one or more other protecting groups present inthe peptide or to cleave the peptide from the resin. Typically, thepeptide will be synthesised using amino acids bearing the appropriateprotecting groups. A person skilled in the art will be able to selectappropriate protecting groups without undue experimentation.

In certain embodiments, the amino acid-comprising conjugation partnerand lipid-containing conjugation partner comprise one or moreunsaturated carbon-carbon bonds in addition to the carbon-carbon doublebond to be reacted. In certain embodiments, the peptide-containingconjugation partner and lipid-containing conjugation partner compriseone or more unsaturated carbon-carbon bonds in addition to thecarbon-carbon double bond to be reacted. Those skilled in the art willappreciate that the selectivity of the thiol for the carbon-carbondouble bond to be reacted in such embodiments may depend on, forexample, the steric and/or electronic environment of the carbon-carbondouble bond relative to the one or more unsaturated carbon-carbon bonds.In certain embodiments, the carbon-carbon double bond to be reacted isactivated relative to any other unsaturated carbon-carbon bonds in theamino acid-comprising conjugation partner and lipid-containingconjugation partner. In certain embodiments, the carbon-carbon doublebond to be reacted is activated relative to any other unsaturatedcarbon-carbon bonds in the peptide-containing conjugation partner andlipid-containing conjugation partner.

In some embodiments, the No-amino group of the amino acid of the aminoacid-comprising conjugation partner comprising the carbon-carbon doublebond or thiol is acylated, for example acetylated. In some embodiments,the method of the present invention may comprise acylating, for exampleacetylating, the No-amino group of the amino acid of the aminoacid-comprising conjugation partner comprising the carbon-carbon doublebond or thiol to be reacted.

Where a peptide-containing conjugation partner has been synthesised bySPPS, acylation may be carried out prior to or after cleavage from theresin. In some embodiments, the amino acid residue of thepeptide-containing conjugation partner bearing the carbon-carbon doublebond or thiol to be reacted is an N-terminal amino acid residue, and themethod comprises acylating the N-terminal amino group prior to cleavingthe peptide.

In some embodiments, the method further comprises acylating the No-aminogroup of the amino acid of the amino acid conjugate or the amino acidresidue of the peptide conjugate to which the lipid-containingconjugation partner is conjugated.

Acylation of the No-amino group of an amino acid may be carried out byreacting an amino acid or peptide with an acylating agent in thepresence of base in a suitable solvent, for example DMF. Non-limitingexamples of acylating agents include acid halides, for example acidchlorides such as acetyl chloride, and acid anhydrides, for exampleacetic anhydride. Such agents maybe commercially available or may beprepared by methods well known in the art. Non-limiting examples ofsuitable bases include triethylamine, diisopropylethylamine,4-methylmorpholine, and the like.

In other embodiments, the synthesising the peptide of thepeptide-containing conjugation partner comprises coupling an amino acidor a peptide comprising an amino acid that is acylated at the No-aminogroup and comprises the carbon-carbon double bond or thiol to be reactedto one or more amino acids and/or one or more peptides.

In some embodiments, the method comprises coupling the amino acid of theamino acid conjugate to an amino acid or a peptide to provide a peptideconjugate. In some embodiments, the method comprises coupling the aminoacid of the amino acid conjugate to an amino acid or peptide bound to asolid phase resin support by SPPS. In some embodiments, the methodcomprises coupling the amino acid of the amino acid conjugate to apeptide bound to a solid phase resin support by SPPS. The method maycomprise synthesising the peptide bound to the solid phase resin supportby SPPS.

In some embodiments, the method further comprises coupling the aminoacid of the amino acid conjugate or an amino acid of the peptideconjugate to one or more amino acids or peptides so as to provide apeptide conjugate comprising one or more EBV LMP2 epitopes. In someembodiments, the peptide to be coupled comprises one or more EBV LMP2epitopes. In other embodiments, one or more EBV LMP2 epitopes is formedon coupling. The coupling may be carried out by SPPS as describedherein.

In some embodiments, the method comprises coupling the amino acid of theamino acid conjugate to a peptide bound to a solid phase resin supportby SPPS so as to provide a peptide conjugate comprising one or more EBVLMP2 epitopes.

In one embodiment, the peptide of the peptide conjugate to be coupled isbound to a solid phase resin support, and the method comprises couplingan amino acid of the peptide conjugate to be coupled to an amino acid ora peptide so as to provide a peptide conjugate comprising one or moreEBV LMP2 epitopes.

In an alternate embodiment, the method comprises coupling an amino acidof the peptide conjugate to an amino acid or peptide bound to a solidphase resin support by SPPS so as to provide peptide conjugatecomprising a peptide epitope.

In some embodiments, the method further comprises coupling an epitope,for example a peptide epitope, to the amino acid conjugate or peptideconjugate. Where the method comprises coupling a peptide epitope, thecoupling may be carried out by SPPS as described herein.

In certain embodiments, the epitope, for example one or more EBV LMP2epitopes, is coupled or bound via a linker group. In certainembodiments, the linker group is an amino sequence, for example asequence of two or more, three or more, or four or more contiguous aminoacids. In certain embodiments, the linker comprises from about 2 to 20,2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 4 to 20, 4 to 18, 4 to 16,4 to 14, 4 to 12, or 4 to 10 amino acids.

It will be appreciate by those skilled in the art that coupling an aminoacid or a peptide to another amino acid or peptide as described hereinmay comprise forming a peptide bond between the No-terminus of the aminoacid or an amino acid of the peptide of one coupling partner and theC-terminus of the amino acid or an amino acid of the peptide of theother coupling partner.

In some embodiments, the method of the present invention comprisessynthesising the amino acid sequence of the peptide of thepeptide-containing conjugation partner by SPPS; and reacting thelipid-containing conjugation partner with the peptide-containingconjugation partner.

In some embodiments, synthesising the amino acid sequence of the peptideof the peptide-containing conjugation partner by SPPS comprises couplingan amino acid or peptide to an amino acid or peptide bound to a solidphase resin support to provide the amino acid sequence of the peptide ora portion thereof. In certain embodiments, the amino acid sequence ofthe entire peptide of the peptide-containing conjugation partner issynthesised by SPPS.

The peptide-containing conjugation partner may be reacted with thelipid-containing conjugation partner while bound to a solid phase resinsupport. Alternatively, the peptide may be cleaved from the solid phaseresin support, and optionally purified, prior to reaction with thelipid-containing conjugation partner.

The peptide conjugate and/or amino acid-comprising conjugation partner,for example a peptide-containing conjugation partner, may comprise oneor more solubilising groups.

The one or more solubilising groups increase the solubility of, forexample, the peptide-containing conjugation partner in polar solvents,such as water. In exemplary embodiments, the solubilising group does notadversely affect the biological activity of the peptide conjugate.

The presence of a solubilising group may be advantageous for formulationand/or administration of the peptide conjugate as a pharmaceuticalcomposition.

In some embodiments, the solubilising group is bound to the peptide ofthe peptide conjugate and/or peptide-containing conjugation partner. Insome embodiments, the solubilising group is bound to the peptide of thepeptide-containing conjugation partner. In some embodiments, the peptideof the peptide conjugate and/or the peptide of the peptide-containingpartner comprises a solubilising group. In some embodiments, the peptideof the peptide-containing partner comprises a solubilising group.

In some embodiments, the solubilising group is bound to the side chainof an amino acid in the peptide chain. In some embodiments, thesolubilising group is bound to the C- or N-terminus of the peptidechain. In some embodiments, the solubilising group is bound between twoamino acid residues in the peptide chain. In some embodiments, thesolubilising group is bound to the No-amino group of one amino acidresidue in the peptide chain and the carboxyl group of another aminoacid residue in the peptide chain.

Examples of suitable solubilising groups include, but are not limitedto, hydrophilic amino acid sequences or polyethylene glycols (PEGs).

In one embodiment, the solubilising group is a hydrophilic amino acidsequence comprising two or more hydrophilic amino acid residues in thepeptide chain. In some embodiments, the solubilising group is an aminoacid sequence comprising a sequence of two or more consecutivehydrophilic amino acid residues in the peptide chain. Such solubilisinggroups may be formed by adding each amino acid of the solubilising groupto the peptide chain by SPPS.

In another embodiment, the solubilising group is a polyethylene glycol.In some embodiments, the polyethylene glycol is bound to the No-aminogroup of one amino acid residue in the peptide chain and the carboxylgroup of another amino acid residue in the peptide chain.

In some embodiments, the polyethylene glycol comprises from about 1 toabout 100, about 1 to about 50, about 1 to about 25, about 1 to about20, about 1 to about 15, about 1 to about 10, about 2 to about 10, orabout 2 to about 4 ethylene glycol monomer units. Methods for couplingpolyethylene glycols to peptides are known.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises an antigen, for example, an antigenicpeptide. In one embodiment, the peptide of the peptide conjugate orpeptide-containing conjugation partner is or comprises an antigen; or anantigen is bound to peptide, optionally via a linker. In someembodiments, the peptide-containing conjugation partner comprises anantigen, for example, an antigenic peptide. In one embodiment, thepeptide of the peptide-containing conjugation partner is or comprises anantigen; or an antigen is bound to peptide, optionally via a linker.

In one embodiment, the antigen comprises a peptide comprising anepitope. In one embodiment, the peptide comprising an epitope is aglycopeptide comprising an epitope. In one embodiment, the antigencomprises a glycopeptide comprising an epitope.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises an epitope. In some embodiments, thepeptide of the peptide conjugate and/or peptide-containing conjugationpartner comprises an epitope. In some embodiments, thepeptide-containing conjugation partner comprises an epitope. In someembodiments, the peptide of the peptide-containing conjugation partnercomprises an epitope.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises two or more EBV LMP2 epitopes, forexample, the peptide of the peptide conjugate and/or peptide-containingconjugation partner comprises two or more EBV LMP2 epitopes.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner is or comprises a glycopeptide comprising one ormore EBV LMP2 epitopes. In some embodiments, the peptide of the peptideconjugate and/or peptide-containing conjugation partner is aglycopeptide. In some embodiments, the peptide conjugate and/orpeptide-containing conjugation partner comprises a glycopeptidecomprising one or more EBV LMP2 epitopes bound to the peptide of thepeptide conjugate and/or peptide-containing conjugation partner. Forexample, the peptide-containing conjugation partner is or comprises aglycopeptide comprising one or more EBV LMP2 epitopes. In someembodiments, the peptide of the peptide-containing conjugation partneris a glycopeptide. In another example, the peptide-containingconjugation partner comprises a glycopeptide comprising one or more EBVLMP2 epitopes bound to the peptide of the peptide-containing conjugationpartner.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises a proteolytic cleavage site. In someembodiments, the peptide of the peptide conjugate and/orpeptide-containing conjugation partner comprises a proteolytic cleavagesite. In some embodiments, the peptide-containing conjugation partnercomprises a proteolytic cleavage site. In some embodiments, the peptideof the peptide-containing conjugation partner comprises a proteolyticcleavage site.

In some embodiments, the peptide of the peptide conjugate and/orpeptide-containing conjugation partner comprises one or more linkergroups. In some embodiments, the peptide of the peptide-containingconjugation partner comprises one or more linker groups.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises a linker group. In some embodiments, thepeptide-containing conjugation partner comprises a linker group.

In some embodiments, the peptide conjugate and/or peptide-containingconjugation partner comprises an epitope bound to the peptide of thepeptide conjugate and/or peptide-containing conjugation partner via alinker group. In some embodiments, the peptide-containing conjugationpartner comprises an epitope bound to the peptide of thepeptide-containing conjugation partner via a linker group.

Examples of linker groups include but are not limited to amino acidsequences (for example, a peptide), polyethylene glycol, alkyl aminoacids, and the like. In some embodiments, the linker is or comprises aproteolytic cleavage site. In some embodiments, the linker is orcomprises a solubilising group.

In some embodiments, the linker is bound between two amino acid residuesin the peptide chain.

In some embodiments, the linker group is bound to the No-amino group ofone amino acid residue in the peptide conjugate and/orpeptide-containing conjugation partner and the carboxyl group of anotheramino acid residue in the peptide-containing conjugation partner. Insome embodiments, the linker group is bound to the No-amino group of oneamino acid residue in the peptide-containing conjugation partner and thecarboxyl group of another amino acid residue in the peptide-containingconjugation partner.

In certain embodiments, the linker group is cleavable in vivo from theamino acids to which it is bound. In certain embodiments, the linkergroup is cleavable by hydrolysis in vivo. In certain embodiments, thelinker group is cleavable by enzymatic hydrolysis in vivo. Linker groupsmay be introduced by any suitable method known in the art.

The method may further comprise coupling an epitope to the amino acid ofthe amino acid conjugate or the peptide of the peptide conjugate. Theepitope may be bound via a linker group, as described above. In someembodiments, the epitope is a peptide epitope. In some embodiments, themethod comprises coupling a glycopeptide comprising an epitope.

It will be appreciated that in certain desirable embodiments, thepeptide conjugates of the invention maintain appropriate uptake,processing, and presentation by antigen presenting cells. Desirably, thelipid-containing conjugate does not interfere with presentation of anyantigenic peptide present in the conjugate by antigen presenting cells.The examples presented herein establish that conjugates of the inventionare presented by antigen presenting cells comparably withnon-conjugated, related peptides.

Confirmation of the identity of the peptides synthesized may beconveniently achieved by, for example, amino acid analysis, massspectrometry, Edman degradation, and the like.

The method of the present invention may further comprise separating theamino acid conjugate from the liquid reaction medium. Alternatively, themethod of the present invention may further comprise separating thepeptide conjugate from the liquid reaction medium. Any suitableseparation methods known in the art may be used, for example,precipitation and filtration. The conjugate may be subsequentlypurified, for example, by HPLC using one or more suitable solvents.

The present invention also relates to amino acid conjugates and peptideconjugates made by the method of the present invention. The conjugatesare as defined in any of the embodiments described herein.

The present invention also relates to a compound of the formula (V),which is an amino acid conjugate.

The present invention also relates to a compound of the formula (V),which is a peptide conjugate.

The peptide conjugates may be pure or purified, or substantially pure.

As used herein “purified” does not require absolute purity; rather, itis intended as a relative term where the material in question is morepure than in the environment it was in previously. In practice thematerial has typically, for example, been subjected to fractionation toremove various other components, and the resultant material hassubstantially retained its desired biological activity or activities.The term “substantially purified” refers to materials that are at leastabout 60% free, preferably at least about 75% free, and most preferablyat least about 90% free, at least about 95% free, at least about 98%free, or more, from other components with which they may be associatedduring manufacture.

The term “α-amino acid” or “amino acid” refers to a molecule containingboth an amino group and a carboxyl group bound to a carbon which isdesignated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

In certain embodiments the peptide-containing conjugation partnercomprises only natural amino acids. The term “naturally occurring aminoacid” refers to any one of the twenty amino acids commonly found inpeptides synthesized in nature, and known by the one letterabbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Yand V.

The term “amino acid analog” or “non-naturally occurring amino acid”refers to a molecule which is structurally similar to an amino acid andwhich can be substituted for an amino acid. Amino acid analogs include,without limitation, compounds which are structurally identical to anamino acid, as defined herein, except for the inclusion of one or moreadditional methylene groups between the amino and carboxyl group (e.g.,a-amino O-carboxy acids), or for the substitution of the amino orcarboxy group by a similarly reactive group (e.g., substitution of theprimary amine with a secondary or tertiary amine, or substitution or thecarboxy group with an ester).

Unless otherwise indicated, conventional techniques of molecularbiology, microbiology, cell biology, biochemistry and immunology, whichare within the skill of the art may be employed in practicing themethods described herein. Such techniques are explained fully in theliterature, such as, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait,ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook ofExperimental Immunology (D. M. Weir & C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,1987); Current Protocols in Molecular Biology (F. M. Ausubel et al.,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY,1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987); andMethods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A.Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).

The term “peptide” and the like is used herein to refer to any polymerof amino acid residues of any length. The polymer can be linear ornon-linear (e.g., branched), it can comprise modified amino acids oramino acid analogs. The term also encompasses amino acid polymers thathave been modified naturally or by intervention, for example, bydisulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other modification or manipulation, for exampleconjugation with labeling or bioactive component.

The inventors have found that the certain peptide conjugates of thepresent invention have immunological activity.

Cell-mediated immunity is primarily mediated by T-lymphocytes.Pathogenic antigens are expressed on the surface of antigen presentingcells (such as macrophages, B-lymphocytes, and dendritic cells), boundto either major histocompatibility MHC Class I or MHC Class IImolecules. Presentation of pathogenic antigen coupled to MHC Class IIactivates a helper (CD4+) T-cell response. Upon binding of the T-cell tothe antigen-MHC II complex, CD4+ T-cells, release cytokines andproliferate.

Presentation of pathogenic antigens bound to MHC Class I moleculesactivates a cytotoxic (CD8+) T-cell response. Upon binding of the T-cellto the antigen-MHC I complex, CD8+ cells secrete perforin and othermediators, resulting in target cell death. Without wishing to be boundby any theory, the applicants believe that in certain embodiments anenhanced response by CD8+ cells is achieved in the presence of one ormore epitopes recognised by CD4+ cells.

Methods to assess and monitor the onset or progression of acell-mediated response in a subject are well known in the art.Convenient exemplary methods include those in which the presence of orthe level of one or more cytokines associated with a cell-mediatedresponse, such as those identified herein, is assessed. Similarly,cell-based methods to assess or monitor the onset and progression of acell-mediated response are amenable to use in the present invention, andmay include cell proliferation or activation assays, including assaystargeted at identifying activation or expansion of one or morepopulations of immune cells, such as T-lymphocytes.

In certain embodiments, methods of the invention elicit both acell-mediated immune response and a humoral response.

The humoral immune response is mediated by secreted antibodies producedby B cells. The secreted antibodies bind to antigens presented on thesurface of invading pathogens, flagging them for destruction.

Again, methods to assess and monitor the onset or progression of ahumoral response are well known in the art. These include antibodybinding assays, ELISA, skin-prick tests and the like.

Without wishing to be bound by theory, the inventors believe that thepeptide conjugates in some embodiments stimulate Toll like receptors(TLRs).

Toll-like receptors (TLRs) are highly conserved pattern recognitionreceptors (PRRs) that recognise pathogen-associated molecular patternsand transmit danger signals to the cell (Kawai, T., Akira, S., Immunity2011, 34, 637-650). TLR2 is a cell-surface receptor expressed on a rangeof different cell types, including dendritic cells, macrophages andlymphocytes (Coffman, R. L., Sher, A., Seder, R. A., Immunity 2010, 33,492-503).

TLR2 recognises a wide range of microbial components includinglipopolysaccharides, peptidoglycans and lipoteichoic acid. It is uniqueamongst TLRs in that it forms heterodimers, with either TLR1 or TLR6;the ability to form complexes with other PRRs may explain the wide rangeof agonists for TLR2 (Feldmann, M., Steinman, L., Nature 2005, 435,612-619). Upon ligand binding and heterodimerisation, signalling takesplace via the MyD88 pathway, leading to NFκB activation and consequentproduction of inflammatory and effector cytokines.

Di- and triacylated lipopeptides derived from bacterial cell-wallcomponents have been extensively studied as TLR2 agonists (Eriksson, E.M. Y., Jackson, D. C., Curr. Prot. and Pept. Sci. 2007, 8, 412-417).Lipopeptides have been reported to promote dendritic cell maturation,causing the up-regulation of co-stimulatory molecules on the cellsurface and enhanced antigen-presentation. Lipopeptides have also beenreported to stimulate macrophages to release cytokines and promote theactivation of lymphocytes including B cells and CD8+ T cells.

In some embodiments, the peptide conjugate has TLR2 agonist activity. Insome embodiments, the peptide conjugate has TLR2 agonist activitycomparable to Pam3CSK4. In some embodiments, the peptide conjugate hasTLR2 agonist activity at least about 50%, about 60%, about 70%, about80%, about 90% that of Pam3CSK4. In some embodiments, for example inembodiments where a modulated immune response is desirable, the peptideconjugate has TLR2 agonist activity less that that of Pam3CSK4. Forexample, the peptide conjugate has TLR2 agonist activity less than about50%, less than about 40%, less than about 30%, less than about 20%, orless than about 10% that of Pam3CSK4.

In some embodiments, the peptide of the peptide conjugate and/orpeptide-containing conjugation partner comprises a serine amino acidresidue adjacent to the amino acid through which the lipid-containingconjugation partner is conjugated to the peptide. In some embodiments,the peptide of the peptide-containing conjugation partner comprises aserine amino acid residue adjacent to the amino acid through which thelipid-containing conjugation partner is conjugated to the peptide. Thepresence of the serine amino acid residue in this position may enhanceTLR2 binding. In some embodiments, the serine amino acid residue isbound to the C-termini of the amino acid through which thelipid-containing conjugation partner is conjugated to the peptide.

As will be appreciated by those skilled in the art on reading thisdisclosure, the peptide conjugate may comprise an epitope, including,for example two or more epitopes. In some embodiments, the epitope is apeptide epitope. A person skilled in the art will appreciate that a widerange of peptide epitopes may be employed in the present invention.

Antigens

It will be appreciated that a great many antigens, for example tumourantigens or antigens from various pathogenic organisms, have beencharacterised and are suitable for use in the present invention, forexample in combination with compositions, vaccines and conjugatescomprising the EBV LMP2 epitopes and peptides specifically recitedherein. All antigens, whether or not presently characterized, that arecapable of eliciting an immune response are contemplated.

The peptides and conjugates of the present invention find application ina wide range of immunotherapies, including but not limited to thetreatment and prevention of conditions or diseases associated with EBV,including but not limited to the treatment and prevention of cancer andneoplastic conditions including Hodgkin's disease, non-Hodgkin'slymphoma, lymphomas, and lymphoepitheliomas including NPC, and thetreatment of viral re-activation during or following immunosuppression,for example in patients who have had bone marrow transplants orhaematopoietic stem cell transplants.

Also contemplated are antigens, particularly EBV LMP2 peptide antigens,comprising one or more amino acid substitutions, such as one or moreconservative amino acid substitutions.

A “conservative amino acid substitution” is one in which an amino acidresidue is replaced with another residue having a chemically similar orderivatised side chain. Families of amino acid residues having similarside chains, for example, have been defined in the art. These familiesinclude, for example, amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Amino acid analogs (e.g.,phosphorylated or glycosylated amino acids) are also contemplated in thepresent invention, as are peptides substituted with non-naturallyoccurring amino acids, including but not limited to N-alkylated aminoacids (e.g. N-methyl amino acids), D-amino acids, 1-amino acids, andγ-amino acids.

Fragments and variants of antigens are also specifically contemplated.

A “fragment” of a peptide, is a subsequence of the peptide that performsa function that is required for the enzymatic or binding activity and/orprovides three dimensional structure of the peptide, such as the threedimensional structure of a polypeptide.

The term “variant” as used herein refers to peptide sequences, includingfor example peptide sequences different from the specifically identifiedsequences, wherein one or more amino acid residues is deleted,substituted, or added. Variants are naturally-occurring variants, ornon-naturally occurring variants. Variants are from the same or fromother species and may encompass homologues, paralogues and orthologues.In certain embodiments, variants of peptides including peptides possessbiological activities that are the same or similar to those of the wildtype peptides. The term “variant” with reference to peptides encompassesall forms of peptides as defined herein.

Those of skill in the art will appreciate that the conjugates of thepresent invention are in certain embodiments particularly suited forstimulating T-cell responses, for example in the treatment of neoplasticdiseases, including cancer. Conjugates, compositions, and vaccines ofthe present invention comprising one or more tumour antigens arecontemplated. It will be appreciated that tumour antigens contemplatedfor use in the preparation of compositions, vaccines, and/or peptideconjugates of the invention will generally comprise one or morepeptides. In certain embodiments of the invention, including for examplepharmaceutical compositions of the invention, one or more additionaltumour antigens may be present, including tumour antigens wherein theone or more tumour antigens does not comprise peptide. Tumour antigensare typically classified as either unique antigens, or shared antigens,with the latter group including differentiation antigens,cancer-specific antigens, and over-expressed antigens. Examples of eachclass of antigens are amenable to use in the present invention.Representative tumour antigens for use in the treatment, for exampleimmunotherapeutic treatment, or vaccination against neoplastic diseasesincluding cancer, are discussed below.

Compounds, vaccines and compositions comprising one or more antigensprepared using those methods of immunisation are specificallycontemplated.

In certain embodiments, the tumour antigen is a peptide-containingtumour antigen, such as a polypeptide tumour antigen or glycoproteintumour antigens. In certain embodiments, the tumour antigen is asaccharide-containing tumour antigen, such as a glycolipid tumourantigen or a ganglioside tumour antigen. In certain embodiments, thetumour antigen is a polynucleotide-containing tumour antigen thatexpresses a polypeptide-containing tumour antigen, for instance, an RNAvector construct or a DNA vector construct, such as plasmid DNA.

Tumour antigens appropriate for the use in the present inventionencompass a wide variety of molecules, such as (a) peptide-containingtumour antigens, including peptide epitopes (which can range, forexample, from 8-20 amino acids in length, although lengths outside thisrange are also common), lipopolypeptides and glycoproteins, (b)saccharide-containing tumour antigens, including poly-saccharides,mucins, gangliosides, glycolipids and glycoproteins, including and (c)polynucleotides that express antigenic polypeptides. Again, thoseskilled in the art will recognise that a tumour antigen present in aconjugate or composition of the present invention will typicallycomprise peptide. However, embodiments of the invention where one ormore conjugates comprises a tumour antigen that does not itself comprisepeptide, but for example is bound to the amino acid-comprising orpeptide-containing conjugation partner, are contemplated. Similarly,compositions of the invention in which one or more tumour antigens thatdoes not itself comprise peptide is present are contemplated.

In certain embodiments, the tumour antigens are, for example, (a) fulllength molecules associated with cancer cells, (b) homologues andmodified forms of the same, including molecules with deleted, addedand/or substituted portions, and (c) fragments of the same, providedsaid fragments remain antigenic or immunogenic. In certain embodiments,the tumour antigens are provided in recombinant form. In certainembodiments, the tumour antigens include, for example, classI-restricted antigens recognized by CD8+ lymphocytes or classII-restricted antigens recognized by CD4+ lymphocytes.

Shared tumour antigens are generally considered to be native, unmutatedsequences that are expressed by tumours due to epigenetic changes thatallow de-repression of developmentally-repressed genes. Accordingly,shared antigens are typically considered preferable to over-expressed ordifferentiation-associated antigens because there is no expression innormal tissues. Also, the same antigens can be targeted in a number ofcancer patients. For example, the cancer-testis antigen NY-ESO-1 ispresent in the majority of patients with many tumours, and a sizeableminority of patients with other tumours. In another example, breastdifferentiation tumour antigens NYBR-1 and NYBR-1.1 are found in aproportion of breast cancer sufferers. Shared tumour antigens thusrepresent an attractive target for development.

The use of shared tumour antigens, such cancer-testis antigens includingNY-ESO-1, CTSP-1, CTSP-2, CTSP-3, CTSP-4, SSX2, and SCP1, and breastcancer antigens NYBR-1 and NYBR-1.1, in combination with peptides orconjugates of the present invention is specifically contemplated herein.

In one exemplary embodiment, the peptide of the invention, for example,the peptide of the peptide-containing conjugation partner or of thepeptide conjugate, comprises one or more epitopes derived from EBV LMP2.Representative epitopes derived from LMP2 are shown in Table 1 below.

TABLE 1 EBV LMP2 epitopes LMP2 residues AA sequence HLALMP2 Long peptide Seq ID No. 51-60 ESNEEPPPPY A1  76 52-60 SNEEPPPPY A11-1  77 71-79 HSDYQPLGT A1 S-1  78 76-84 PLGTQDQSL A2 S-1  79 76-85PLGTQDQSLY A1 S-1  80 76-85 PLGTQDQSLY A3 S-1  80 77-85 LGTQDQSLY A1 S-1 81 78-86 GTQDQSLYL A2 S-1  82 78-86 GTQDQSLYL A11 S-1  83 78-87GTQDQSLYLG A11 S-1  84 82-90 QSLYLGLQH A3 S-1  85 83-91 SLYLGLQHD A2S-1, S-2  86 83-91 SLYLGLQHD A3 S-1, S-2  86 87-96 GLQHDGNDGL A2S-1, S-2  87  92-101 GNDGLPPPPY A1 S-2  88  95-103 GLPPPPYSP A2 S-2  89 95-104 GLPPPPYSPR A3 S-2  90  95-104 GLPPPPYSPR A11 S-2  90 103-111PRDDSSQHIY A1 S-2  91 104-112 RDDSSQHIY A1 S-2  92 110-118 HIYEEAGRG A32-1  93 350-358 ILLARLFLY A3/A8/A29 5-1, 5-2, 5-4, 5-5  94 340-350SSCSSCPLSKI A11 5-1, 5-2, 5-3, 5-4, 5-5  95 329-337 LLWTLVVLL A25-1, 5-2, 5-3, 5-5  96 356-364 FLYALALLL A2 5-1, 5-2, 5-4, 5-5  97426-434 CLGGLLTMV A2 6-1  98 257-265 LIVDAVLQL A2 4-1  99 453-461LTAGFLIFL A2 6-1 100 243-251 TVCGGIMFL A2 4-1 101

In one specifically contemplated embodiment, the peptide of theinvention, for example, the isolated, purified, or recombinant peptideor the peptide of the peptide-containing conjugation partner or of thepeptide conjugate, comprises, consists essentially of, or consists of anamino acid sequence selected from the group consisting of 8 or morecontiguous, 10 or more contiguous, 12 or more contiguous, 15 or morecontiguous, 20 or more contiguous, or 25 or more contiguous amino acidsfrom any one of SEQ ID NOs: 1 to 101, for example, from any one of SEQID NOs: 1 to 93, including for example any one of SEQ ID NOs: 1 to 75.

In various embodiments, the peptide comprises more that one amino acidsequence selected from the group consisting of any one of SEQ ID NOs: 1to 101. In one embodiment, the peptide comprises one or more amino acidsequences selected from the group consisting of SEQ ID NOs: 76-101, orfrom the group consisting of SEQ ID NOs: 76-93.

In one specifically contemplated embodiment, the peptide of theinvention, for example, the isolated, purified, or recombinant peptideor the peptide of the peptide-containing conjugation partner or of thepeptide conjugate, comprises, consists essentially of, or consists of anamino acid sequence selected from the group consisting of 8 or morecontiguous, 10 or more contiguous, 12 or more contiguous, 15 or morecontiguous, 20 or more contiguous, or 25 or more contiguous amino acidsfrom any one of the sequences depicted in Table 2 below.

TABLE 2 LMP2 long peptides LMP2 SEQ ID NAME residues SEQUENCE NO. S-169-96 DRHSDYQPLGTQDQSLYLGLQHDGNDGL  5 S-2  83-115SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA 10 S-3 72-96 SDYQPLGTQDQSLYLGLQHDGNDGL15 S-4 69-115 DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPP 20 PYSPRDDSSQHIYEEA 5-1329-364 LLWTLVVLLICSSCSSCPLSKILLARLFLY 25 ALALLL 5-2 327-366LMLLWTLVVLLICSSCSSCPLSKILLARLFLY 30 ALALLLLA 5-3 327-352LMLLWTLVVLLICSSCSSCPLSKILL 35 5-4 336-366LLICSSCSSCPLSKILLARLFLYALALLLLA 40 5-5 319-375LNLTTMFLLMLLWTLVVLLICSSCSSCPLSK 45 ILLARLFLYALALLLLASALIAGGSI 5-6325-368 FLLMLLWTLVVLLICSSCSSCPLSKILLARL 50 FLYALALLLLASA 4-1 219-269LQGIYVLVMLVLLILAYRRRWRRLTVCGGIM 55 FLACVLVLIVDAVLQLSPLL 6-1 415-465SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWL 60 TVMSNTLLSAWILTAGFLIFLIGFA 1-1 52-98SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSL 65 YLGLQHDGNDGLPP 2-1  92-142GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNP 70 VCLPVIVAPYLFWLAAIAAS 3-1 137-186AAIAASCFTASVSTVVTATGLALSLLLLAAV 75 ASSYAAAQRKLLTPVTVLT

Similarly, the prostate vaccine Sipuleucel-T (APC8015, Provenge™), whichcomprises the antigen prostatic acid phosphatase (PAP), is present in95% of prostate cancer cells. At least in part due to this potential forefficacy in a significant proportion of prostate cancer sufferers,Sipuleucel-T was approved by the FDA in 2010 for use in the treatment ofasymptomatic, hormone-refractory prostate cancer. The use of PAP antigenin conjugates of the present invention is specifically contemplated inthe present invention.

Unique antigens are considered to be those antigens that are unique toan individual or are shared by a small proportion of cancer patients,and typically result from mutations leading to unique protein sequences.Representative examples of unique tumour antigens include mutated Rasantigens, and mutated p53 antigens. As will be appreciated by thoseskilled in the art having read this specification, the methods of thepresent invention enable the ready preparation of conjugates comprisingone or more unique tumour antigens, for example to elicit specificT-cell responses to one or more unique tumour antigens, for example inthe preparation of patient-specific therapies.

Accordingly, representative tumour antigens include, but are not limitedto, (a) antigens such as RAGE, BAGE, GAGE and MAGE family polypeptides,for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5,MAGE-6, and MAGE-12 (which can be used, for example, to addressmelanoma, lung, head and neck, NSCLC, breast, gastrointestinal, andbladder tumours), (b) mutated antigens, for example, p53 (associatedwith various solid tumours, for example, colorectal, lung, head and neckcancer), p21/Ras (associated with, for example, melanoma, pancreaticcancer and colorectal cancer), CDK4 (associated with, for example,melanoma), MUM1 (associated with, for example, melanoma), caspase-8(associated with, for example, head and neck cancer), CIA 0205(associated with, for example, bladder cancer), HLA-A2-R1701, betacatenin (associated with, for example, melanoma), TCR (associated with,for example, T-cell non-Hodgkins lymphoma), BCR-abl (associated with,for example, chronic myelogenous leukemia), triosephosphate isomerase,MA 0205, CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example,Galectin 4 (associated with, for example, colorectal cancer), Galectin 9(associated with, for example, Hodgkin's disease), proteinase 3(associated with, for example, chronic myelogenous leukemia), Wilm'stumour antigen-1 (WT 1, associated with, for example, variousleukemias), carbonic anhydrase (associated with, for example, renalcancer), aldolase A (associated with, for example, lung cancer), PRAME(associated with, for example, melanoma), HER-2/neu (associated with,for example, breast, colon, lung and ovarian cancer), alpha-fetoprotein(associated with, for example, hepatoma), KSA (associated with, forexample, colorectal cancer), gastrin (associated with, for example,pancreatic and gastric cancer), telomerase catalytic protein, MUC-1(associated with, for example, breast and ovarian cancer), G-250(associated with, for example, renal cell carcinoma), p53 (associatedwith, for example, breast, colon cancer), and carcinoembryonic antigen(associated with, for example, breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer), (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, for example, melanoma), (e) prostateassociated antigens such as PAP, prostatic serum antigen (PSA), PSMA,PSH-P1, PSM-P1, PSM-P2, associated with for example, prostate cancer,(f) immunoglobulin idiotypes (associated with myeloma and B celllymphomas, for example), and (g) other tumour antigens, such aspolypeptide- and saccharide-containing antigens including (i)glycoproteins such as sialyl Tn and sialyl Le.sup.x (associated with,for example, breast and colorectal cancer) as well as various mucins;glycoproteins are coupled to a carrier protein (for example, MUC-1 arecoupled to KLH); (ii) lipopolypeptides (for example, MUC-1 linked to alipid moiety); (iii) polysaccharides (for example, Globo H synthetichexasaccharide), which are coupled to a carrier proteins (for example,to KLH), (iv) gangliosides such as GM2, GM12, GD2, GD3 (associated with,for example, brain, lung cancer, melanoma), which also are coupled tocarrier proteins (for example, KLH).

Other representative tumour antigens amenable to use in the presentinvention include TAG-72, (See, e.g., U.S. Pat. No. 5,892,020; humancarcinoma antigen (See, e.g., U.S. Pat. No. 5,808,005); TP1 and TP3antigens from osteocarcinoma cells (See, e.g., U.S. Pat. No. 5,855,866);Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells (See, e.g.,U.S. Pat. No. 5,110,911); KC-4 antigen from human prostrateadenocarcinoma (See, e.g., U.S. Pat. No. 4,743,543); a human colorectalcancer antigen (See, e.g., U.S. Pat. No. 4,921,789); CA125 antigen fromcystadenocarcinoma (See, e.g., U.S. Pat. No. 4,921,790); DF3 antigenfrom human breast carcinoma (See, e.g., U.S. Pat. Nos. 4,963,484 and5,053,489); a human breast tumour antigen (See, e.g., U.S. Pat. No.4,939,240); p97 antigen of human melanoma (See, e.g., U.S. Pat. No.4,918,164); carcinoma or orosomucoid-related antigen (CORA) (See, e.g.,U.S. Pat. No. 4,914,021); T and Tn haptens in glycoproteins of humanbreast carcinoma, MSA breast carcinoma glycoprotein; MFGM breastcarcinoma antigen; DU-PAN-2 pancreatic carcinoma antigen; CA125 ovariancarcinoma antigen; YH206 lung carcinoma antigen, Alphafetoprotein (AFP),hepatocellular carcinoma antigen; Carcinoembryonic antigen (CEA); bowelcancer antigen; Epithelial tumour antigen (ETA); breast cancer antigen;Tyrosinase; the raf oncogene product; gp75; gp100; EBV-LMP 1 & 2;EBV-EBNA 1, 2 & 3C; HPV-E4, 6, 7; C017-1A; GA733; gp72; p53; proteinase3; telomerase; and melanoma gangliosides. These and other tumourantigens, whether or not presently characterized, are contemplated foruse in the present invention.

In certain embodiments, the tumour antigens are derived from mutated oraltered cellular components. Representative examples of altered cellularcomponents include, but are not limited to ras, p53, Rb, altered proteinencoded by the Wilms' tumour gene, ubiquitin, mucin, protein encoded bythe DCC, APC, and MCC genes, as well as receptors or receptor-likestructures such as neu, thyroid hormone receptor, platelet derivedgrowth factor (PDGF) receptor, insulin receptor, epidermal growth factor(EGF) receptor, and the colony stimulating factor (CSF) receptor.

Polynucleotide-containing antigens used in the present invention includepolynucleotides that encode polypeptide tumour antigens such as thoselisted above. In certain embodiments, the polynucleotide-containingantigens include, but are not limited to, DNA or RNA vector constructs,such as plasmid vectors (e.g., pCMV), which are capable of expressingpolypeptide tumour antigens in vivo.

The present invention also contemplates the preparation of conjugatescomprising viral antigens that are capable of stimulating T-cell toelicit effective anti-viral immunity in patients who are or have beenimmunosuppressed, for example patients who have had bone marrowtransplants, haematopoietic stem cell transplants, or are otherwiseundergoing immunosuppression.

Similarly, antigens derived from viruses associated with increasedincidence of cancer, or that are reported to be cancer-causing, such ashuman papillomavirus, hepatitis A virus, and hepatitis B virus, arecontemplated for use in the present invention.

For example, in certain embodiments, the tumour antigens include, butare not limited to, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK,MYL-RAR, Epstein Barr virus antigens, human papillomavirus (HPV)antigens, including E6 and E7, hepatitis B and C virus antigens, humanT-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3,c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16,TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125,CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

The above-listed or referenced antigens are exemplary, not limiting, ofthe present invention.

The present invention also relates to pharmaceutical compositioncomprising an effective amount of a peptide conjugate of the presentinvention or a pharmaceutically acceptable salt or solvent thereof, anda pharmaceutically acceptable carrier.

The present invention relates to a pharmaceutical composition comprisingan effective amount of a peptide of the present invention or apharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable carrier.

The pharmaceutical compositions may comprise an effective amount of twoor more peptides of the invention, two or more peptide conjugates of theinvention, or one more peptides of the invention and one or more peptideconjugates of the invention in combination.

The term “pharmaceutically acceptable carrier” refers to a carrier(adjuvant or vehicle) that may be administered to a subject togetherwith the peptide or peptide conjugate of the present invention, or apharmaceutically acceptable salt or solvent thereof, and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers that may be used in thecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α—, β—, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery. Oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agents,which are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions.

The compositions are formulated to allow for administration to a subjectby any chosen route, including but not limited to oral or parenteral(including topical, subcutaneous, intramuscular and intravenous)administration.

For example, the compositions may be formulated with an appropriatepharmaceutically acceptable carrier (including excipients, diluents,auxiliaries, and combinations thereof) selected with regard to theintended route of administration and standard pharmaceutical practice.For example, the compositions may be administered orally as a powder,liquid, tablet or capsule, or topically as an ointment, cream or lotion.Suitable formulations may contain additional agents as required,including emulsifying, antioxidant, flavouring or colouring agents, andmay be adapted for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release.

The compositions may be formulated to optimize bioavailability,immunogenicity, or to maintain plasma, blood, or tissue concentrationswithin the immunogenic or therapeutic range, including for extendedperiods. Controlled delivery preparations may also be used to optimizethe antigen concentration at the site of action, for example.

The compositions may be formulated for periodic administration, forexample to provide continued exposure. Strategies to elicit a beneficialimmunological response, for example those that employ one or more“booster” vaccinations, are well known in the art, and such strategiesmay be adopted.

The compositions may be administered via the parenteral route. Examplesof parenteral dosage forms include aqueous solutions, isotonic saline or5% glucose of the active agent, or other well-known pharmaceuticallyacceptable excipients. Cyclodextrins, for example, or other solubilisingagents well-known to those familiar with the art, can be utilized aspharmaceutical excipients for delivery of the therapeutic agent.

Examples of dosage forms suitable for oral administration include, butare not limited to tablets, capsules, lozenges, or like forms, or anyliquid forms such as syrups, aqueous solutions, emulsions and the like,capable of providing a therapeutically effective amount of thecomposition. Capsules can contain any standard pharmaceuticallyacceptable materials such as gelatin or cellulose. Tablets can beformulated in accordance with conventional procedures by compressingmixtures of the active ingredients with a solid carrier and a lubricant.Examples of solid carriers include starch and sugar bentonite. Activeingredients can also be administered in a form of a hard shell tablet ora capsule containing a binder, e.g., lactose or mannitol, a conventionalfiller, and a tabletting agent.

Examples of dosage forms suitable for transdermal administrationinclude, but are not limited, to transdermal patches, transdermalbandages, and the like.

Examples of dosage forms suitable for topical administration of thecompositions include any lotion, stick, spray, ointment, paste, cream,gel, etc., whether applied directly to the skin or via an intermediarysuch as a pad, patch or the like.

Examples of dosage forms suitable for suppository administration of thecompositions include any solid dosage form inserted into a bodilyorifice particularly those inserted rectally, vaginally and urethrally.

Examples of dosage of forms suitable for injection of the compositionsinclude delivery via bolus such as single or multiple administrations byintravenous injection, subcutaneous, subdermal, and intramuscularadministration or oral administration.

Examples of dosage forms suitable for depot administration of thecompositions and include pellets of the peptides or peptide conjugatesor solid forms wherein the peptides or peptide conjugates are entrappedin a matrix of biodegradable polymers, microemulsions, liposomes or aremicroencapsulated.

Examples of infusion devices for the compositions include infusion pumpsfor providing a desired number of doses or steady state administration,and include implantable drug pumps.

Examples of implantable infusion devices for compositions include anysolid form in which the peptides or peptide conjugates are encapsulatedwithin or dispersed throughout a biodegradable polymer or synthetic,polymer such as silicone, silicone rubber, silastic or similar polymer.

Examples of dosage forms suitable for transmucosal delivery of thecompositions include depositories solutions for enemas, pessaries,tampons, creams, gels, pastes, foams, nebulised solutions, powders andsimilar formulations containing in addition to the active ingredientssuch carriers as are known in the art to be appropriate. Such dosageforms include forms suitable for inhalation or insufflation of thecompositions, including compositions comprising solutions and/orsuspensions in pharmaceutically acceptable, aqueous, or organicsolvents, or mixture thereof and/or powders. Transmucosal administrationof the compositions may utilize any mucosal membrane but commonlyutilizes the nasal, buccal, vaginal and rectal tissues. Formulationssuitable for nasal administration of the compositions may beadministered in a liquid form, for example, nasal spray, nasal drops, orby aerosol administration by nebulizer, including aqueous or oilysolutions of the polymer particles. Formulations may be prepared asaqueous solutions for example in saline, solutions employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebio-availability, fluorocarbons, and/or other solubilising or dispersingagents known in the art.

Examples of dosage forms suitable for buccal or sublingualadministration of the compositions include lozenges, tablets and thelike. Examples of dosage forms suitable for opthalmic administration ofthe compositions include inserts and/or compositions comprisingsolutions and/or suspensions in pharmaceutically acceptable, aqueous, ororganic solvents.

Examples of formulations of compositions, including vaccines, may befound in, for example, Sweetman, S. C. (Ed.). Martindale. The CompleteDrug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483pp.; Aulton, M. E. (Ed.) Pharmaceutics. The Science of Dosage FormDesign. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and, Ansel, H.C., Allen, L. V. and Popovich, N. G. Pharmaceutical Dosage Forms andDrug Delivery Systems, 7th Ed., Lippincott 1999, 676 pp. Excipientsemployed in the manufacture of drug delivery systems are described invarious publications known to those skilled in the art including, forexample, Kibbe, E. H. Handbook of Pharmaceutical Excipients, 3rd Ed.,American Pharmaceutical Association, Washington, 2000, 665 pp. TheUnited States Pharmacopeia also provides examples of modified-releaseoral dosage forms, including those formulated as tablets or capsules.See, for example, The United States Pharmacopeia 23/National Formulary18, The United States Pharmacopeial Convention, Inc., Rockville Md.,1995 (hereinafter “the USP”), which also describes specific tests todetermine the drug release capabilities of extended-release anddelayed-release tablets and capsules. The USP test for drug release forextended-release and delayed-release articles is based on drugdissolution from the dosage unit against elapsed test time. Descriptionsof various test apparatus and procedures may be found in the USP.Further guidance concerning the analysis of extended release dosageforms has been provided by the F.D.A. (See Guidance for Industry.Extended release oral dosage forms: development, evaluation, andapplication of in vitro/in vivo correlations. Rockville, Md.: Center forDrug Evaluation and Research, Food and Drug Administration, 1997).

While the composition may comprise one or more extrinsic adjuvants,advantageously in some embodiments this is not necessary. In someembodiments, the peptide conjugate comprises an epitope and is selfadjuvanting.

The present invention provides a method of vaccinating or eliciting animmune response in a subject comprising administering to the subject aneffective amount of a peptide conjugate or peptide of the presentinvention. The present invention also relates to use of a peptideconjugate or peptide of the invention for vaccinating or eliciting animmune response in a subject, and to use of a peptide conjugate or apeptide of the invention in the manufacture of a medicament forvaccinating or eliciting an immune response in a subject.

The present invention also provides a method of vaccinating or elicitingan immune response in a subject comprising administering to the subjectan effective amount of the pharmaceutical composition of the presentinvention. The present invention also relates to use of a pharmaceuticalcomposition of the invention for vaccinating or eliciting an immuneresponse in a subject, and to the use of one or more peptides of thepresent invention or one or more peptide conjugates of the presentinvention in the manufacture of a medicament for vaccinating oreliciting an immune response in a subject.

The present invention provides a method of eliciting an immune responsein a subject comprising administering to the subject an effective amountof a peptide of the present invention. The present invention alsorelates to use of a conjugate of the invention for eliciting an immuneresponse, and to use of a peptide conjugate of the invention in themanufacture of a medicament for eliciting an immune response in asubject.

The present invention provides a method of vaccinating a subjectcomprising administering to the subject an effective amount of a peptideof the present invention. The present invention also relates to use of aconjugate of the invention for eliciting an immune response, and to useof a peptide conjugate of the invention in the manufacture of amedicament for eliciting an immune response in a subject.

The administration or use of one or more peptides of the presentinvention and/or one or more peptide conjugates of the presentinvention, for example one or more peptide in together with one or morepeptide conjugates, for vaccinating or eliciting an immune response inthe subject is contemplated herein.

Where two or more peptides, two or more peptide conjugates, or one ormore peptides and one or more peptide conjugates are administered orused, the two or more peptides, two or more peptide conjugates, or oneor more peptides and one or more peptide conjugates may be administeredor used simultaneously, sequentially, or separately.

A “subject” refers to a vertebrate that is a mammal, for example, ahuman. Mammals include, but are not limited to, humans, farm animals,sport animals, pets, primates, mice and rats.

An “effective amount” is an amount sufficient to effect beneficial ordesired results including clinical results. An effective amount can beadministered in one or more administrations by various routes ofadministration.

The effective amount will vary depending on, among other factors, thedisease indicated, the severity of the disease, the age and relativehealth of the subject, the potency of the compound administered, themode of administration and the treatment desired. A person skilled inthe art will be able to determine appropriate dosages having regard tothese any other relevant factors.

The efficacy of a composition can be evaluated both in vitro and invivo. For example, the composition can be tested in vitro or in vivo forits ability to induce a cell-mediated immune response. For in vivostudies, the composition can be fed to or injected into an animal (e.g.,a mouse) and its effects on eliciting an immune response are thenassessed. Based on the results, an appropriate dosage range andadministration route can be determined.

The composition may be administered as a single dose or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule.

In certain embodiments, eliciting an immune response comprises raisingor enhancing an immune response. In exemplary embodiments, eliciting animmune response comprises eliciting a humoral and a cell mediatedresponse.

In certain embodiments, eliciting an immune response provides immunity.

The immune response is elicited for treating a disease or condition. Aperson skilled in the art will appreciate that the peptides and peptideconjugates described herein are useful for treating a variety ofdiseases and conditions associated with EBV, including one or morediseases or conditions selected from EBV-associated neoplasticconditions, including B and T cell non-Hodgkin's lymphomas, Hodgkin'sdisease, and lymphoepithelioma-like carcinomas, including but notlimited to nasopharyngeal carcinoma (NPC).

In some embodiments, the disease or condition is an infectious disease,cancer, or viral re-activation post-bone marrow transplant or followinginduction of profound immunosuppression for any other reason.

The term “treatment”, and related terms such as “treating” and “treat”,as used herein relates generally to treatment, of a human or a non-humansubject, in which some desired therapeutic effect is achieved. Thetherapeutic effect may, for example, be inhibition, reduction,amelioration, halt, or prevention of a disease or condition.

The compositions may be used to elicit systemic and/or mucosal immunity.Enhanced systemic and/or mucosal immunity may be reflected in anenhanced TH1 and/or TH2 immune response. The enhanced immune responsemay include an increase in the production of IgG1 and/or IgG2a and/orIgA.

The invention consists in the foregoing and also envisages constructionsof which the following gives examples only and in no way limit the scopethereof.

EXAMPLES Example 1. Preparation of Conjugates 200, 20, 22, and 26

1.1 General Details

Commercially available starting materials are purchased from AcrosOrganics, Ajax Finechem, Alfa Aesar, CEM, GL-Biochem, Merck, NOVABiochem, Sigma Aldrich and TCI and are used as supplied. Dried solventsare prepared through distillation under N₂ or argon atmosphere.Tetrahydrofuran (THF) is freshly distilled over sodium/benzophenoneketyl. Methanol (MeOH) and toluene are freshly distilled over calciumhydride. Yields refer to chromatographically and spectroscopically (¹HNMR) homogenous materials unless otherwise stated.

Thin layer chromatography (TLC) is performed on Merck Kieselgel F₂₅₄ 200μm silica plates. Ultraviolet light is used as a visualising agent andthe general developing agents of potassium permanganate in an aqueousbasic solution and vanillin in an ethanolic solution. Specificdeveloping agents used are ethanolic solutions of ninhydrin with acidfor the identification of primary amines. Heating is applied when usingany developing agent. Silica gel (0.063-0.100 mm) is used for flashcolumn chromatography.

Nuclear magnetic resonance (NMR) spectra are acquired at roomtemperature in CDCl₃ or D₂O on a Bruker DRX400 spectrometer operating at400 MHz for ¹H nuclei and 100 MHz for ¹³C nuclei. Reference peaks for ¹Hand ¹³C spectra are respectively set to 60.00 and δ 77.0 for CDCl₃ andδ4.79 for ¹H spectra in D₂O. NMR data are reported in values of chemicalshift as parts per million (ppm) on the δ scale, and coupling constantsin hertz (Hz). Multiplicities are reported as s=singlet, d=doublet,t=triplet, q=quartet, dd=doublet of doublets, dt=doublet of triplets,tt=triplet of triplets, dq=doublet of quartets, dqn=doublet of quintets,sx=sextet, br s=broad singlet, and m=multiplet. The assignment of C_(q)is used to denote a quaternary carbon.

High resolution mass spectra are obtained on a Bruker microOTOF-Q IImass spectrometer at a nominal resolution of 5000. Analyticalhigh-performance liquid chromatography (HPLC) and liquidchromatography-mass spectrometry (LC-MS) chromatograms are acquired oneither a Dionex UltiMate 3000 HPLC system with a Finnigan Surveyor MSQPlus mass spectrometer or an Agilent 1120 Compact LC system with aHewlett Packard Series 1100 MSD mass spectrometer. Analytical reversephase (RP) HPLC is performed using the MeCN/H₂O+0.1% TFA solvent system.

Semipreparative RP HPLC is performed on a Dionex UltiMate 3000 HPLCsystem using the MeCN/H₂O+0.1% TFA solvent system. Microwave reactionsare performed using a CEM Liberty Automated Microwave system.

1.2 General Method for Peptide Chain Elongation

Manual Synthesis Method

Swelled peptide-resin is treated with 20% v/v piperidine in DMF (5.0 mL)and shaken for 20 min at r.t. The solution is drained and the resinwashed with DMF (×2) and DCM (×2). A coupling mixture of Fmoc-AA-OH (2.0eq.), HBTU (2.0 eq) and iPr₂NEt (4.0 eq.) in DMF (1 mL) is added and theresin shaken for 1 hr. Resin is drained and washed again. The procedureis repeated for the remaining residues in the sequence.

Automated Synthesis Method (Standard, 0.2 Mmol Scale)

Peptide-resin is transferred to the reaction vessel of a Tributeautomated peptide synthesiser. Automated synthesis is undertaken withcycles of Fmoc deprotection and Fmoc-AA-OH coupling steps. Deprotectionis undertaken by addition of 20% v/v piperidine in DMF (6.0 mL) andagitation (2×7 min). Following resin drainage and DMF washing (4 mL×3),a coupling step is performed with 5 eq. Fmoc-AA-OH dissolved in HBTU(0.24 mM, in DMF, 4 mL). 2 M N-methylmorpholine (NMM) in DMF (4 mL) isutilised in the base-addition step. Coupling proceeded for 1 hr. AfterDMF washing steps, the next cycle of deprotection and couplingcommenced, repeating until all amino acids are coupled.

Procedure for Coupling of Cysteine Derivatives (0.1 Mmol Scale)

Peptide-resin is swelled in 1:1 CH₂Cl₂:DMF for 30 min, then drained. Acoupling mixture of a Cys amino acid (0.2 mmol, 2 eq.), BOP (0.4 mmol, 4eq.) and HOBt.H₂O (0.4 mmol, 4 eq.) is dissolved in 1:1 CH₂Cl₂:DMF (2mL). 2,4,6-collidine (0.4 mmol, 4 eq.) is then added and the resultantsolution added to the peptide-resin. The resin is agitated for 1 hr, oruntil ninhydrin test indicated no free amines. The resin is thendrained, washed with DMF (2×) and CH₂Cl₂ (2×), and dried.

Ninhydrin Test Procedure

A small portion of resin is taken, washed with CH₂Cl₂ and allowed todry. 1 drop each of solutions of 5% v/v ninhydrin in EtOH, 80% w/vphenol in EtOH and 2% v/v KCN in pyridine are added to the resin and themixture heated at 90° C. for 2 minutes. Blue-coloured beads and solutionindicated the presence of free primary amines, while a yellow colourindicated no free amino groups present.

1.3 Preparation of Amino Add Conjugate 200

N-Fluorenylmethoxycarbonyl-[R]-cysteine

Fmoc-Cys(Trt)-OH (1.0 g, 1.7 mmol) is dissolved in CH₂Cl₂ (50 mL). TFA(1.5 mL, 19.6 mmol) and iPr₃SiH (0.75 mL) are added, causing thesolution to turn yellow. The solution is agitated for 2 hrs at roomtemperature, at which point the solution turns colourless. The mixtureis basified to pH 9 by addition of Na₂CO₃.H₂O and washed with EtOAc. Thesolution is acidified with 10M HCl, extracted with EtOAc andconcentrated in vacuo to give a white powder and a pink residue. Thepowder and residue are dissolved in 4:1 MeCN:H₂O and lyophilised, givinga crude pink-white powder (424 mg, crude yield 73.1%). This crudeproduct is carried through to the thiol-ene reactions described below.

(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((2-(palmitoyloxy)ethyl)thio)propanoicadd (200)

Thermal Initiation (Li, J. Dong, S. et al. Chemistry as an ExpandingResource in Protein Science: Fully Synthetic and Fully Active HumanParathyroid Hormone-Related Protein (1-141). Angewandte ChemieInternational Edition 2012, 51 (49), 12263-12267).

Fmoc-Cys-OH (100 mg, 0.29 mmol), vinyl palmitate (476 μL, 1.5 mmol) andAIBN (9.6 mg, 59 μmol) are dissolved in degassed 1,2-dichloroethane (3mL). The reaction mixture is then heated under reflux (90° C.) for 24hr, after which TLC indicated complete consumption of Fmoc-Cys-OH. Thesolution is then allowed to cool to r.t. The solvent is removed underreduced pressure. Presence of the desired product 200 in the crudereaction mixture is confirmed by mass spectrometry.

Photo-Initiation

Fmoc-Cys-OH (100 mg, 0.29 mmol) is dissolved in degassed, anhydrous DMF(500 μL). Vinyl palmitate (90 μL, 0.3 mmol) and DMPA (5.0 mg, 20 μmol)are dissolved in degassed CH₂Cl₂ (200 μL). The two solutions arecombined and the resultant mixture irradiated for 6 hr (365 nm UV) in astandard photochemical apparatus. When no further change in the reactionmixture can be observed by TLC, solvent is removed under reducedpressure. The crude product is purified by silica gel flashchromatography (3:1 EtOAc:n-hexanes+2% AcOH), followed by lyophilizationfrom 1:1 H2O:MeCN+0.1% TFA to afford the title compound as a powderywhite solid (24 mg, 13%). Structure of the desired product 200 isconfirmed by mass spectrometry.

1.4 Preparation of Peptide Conjugates 20, 22, and 26

Peptides

[SEQ ID NO: 102] AcN-Cys-Ser-Lys-Lys-Lys-Lys-Asp-Arg-His-Ser-Asp-Tyr-Gln-Pro-Leu-Gly-Thr-Gln-Asp-Gln-Ser-Leu-Tyr-Leu-Gly-Leu-Gln-His-Asp-Gly-Asn-Asp-Gly-Leu-OH 25

To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g loading, 0.2mmol scale) pre-swelled in 1:1 CH₂Cl₂:DMF is added a coupling mixture ofFmoc-L-Val-O—CH₂-phi-OCH₂—CH₂—COOH (155.2 mg, 0.3 mmol), HBTU (113.8 mg,0.3 mmol) and iPr₂NEt (104 μL, 0.6 mmol) in DMF (3 mL). The resin isshaken for 2 hrs at r.t., after which a ninhydrin test is performed tomonitor complete coupling. The Fmoc-Val amino group is then deprotectedby treatment of the resin with 20% v/v piperidine in DMF (5 mL) for 20mins at r.t. The resin is transferred to a Tribute automated peptidesynthesiser. Chain elongation up to and including the Ser residue isperformed using the general automated coupling method. Coupling of theFmoc-Cys(Trt)-OH residue is performed manually with addition of amixture of Fmoc-Cys(Trt)-OH (235 mg, 0.4 mmol), BOP (360 mg, 0.8 mmol),HOBt.H₂O (120 mg, 0.8 mmol) and 2,4,6-collidine (120 μL, 0.8 mmol) in1:1 CH₂Cl₂:DMF (2 mL). The resin is shaken for 1 hr at r.t., after whicha ninhydrin test is performed to monitor complete coupling. Final Fmocdeprotection is accomplished by treatment of the resin with 20% v/vpiperidine in DMF (5 mL) for 20 mins at r.t.

After Fmoc deprotection, N-acetylation is performed by adding aceticanhydride (50 μL) and iPr₂NEt (50 μL) in DMF (3 mL) to the resin. Theresin is then shaken for 30 min at r.t., after which a ninhydrin test isperformed to ensure no remaining free amines. The resin is drained,washed with DMF and CH₂Cl₂ and air dried. A cleavage cocktail of TFA:H2O:DODT:iPr₃SiH (94:2.5:2.5:1% v/v, 10.0 mL) is added to the dry resinand the mixture shaken for 4 hr at r.t. The cleavage cocktail is thentreated with cold diethyl ether to precipitate the crude peptide, whichis centrifuged at 4000 rpm for 5 minutes. The supernatant is discardedand the pellet washed with diethyl ether, before repeating the spinningstep. The ether phase is then discarded and the peptide dried with N₂flow. The crude peptide is then lyophilised from H₂O+0.1% TFA. The crudeproduct is carried through to the thiol-ene reaction step outlinedbelow.

To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g loading, 0.2mmol scale) pre-swelled in 1:1 CH₂Cl₂:DMF is added a coupling mixture ofFmoc-Rink-Amide-OH (216 mg, 0.4 mmol), HBTU (151.8 mg, 0.4 mmol) andiPraNEt (140 μL, 0.8 mmol) in DMF (2 mL). The resin is shaken for 1 hrat r.t., after which a ninhydrin test is performed to establish completecoupling. The linker amino group is then deprotected by treatment of theresin with 20% v/v piperidine in DMF (5 mL) for 20 mins at r.t. Theresin is transferred to a Tribute automated peptide synthesiser. Chainelongation up to and including the Ser residue is performed using thegeneral automated coupling method. Coupling of the Cys residue isperformed manually with addition of a mixture of Fmoc-Cys(Trt)-OH (235mg, 0.4 mmol), BOP (360 mg, 0.8 mmol), HOBt.H₂O (120 mg, 0.8 mmol) and2,4,6-collidine (120 μL, 0.8 mmol) in 1:1 CH₂Cl₂:DMF (2 mL). The resinis shaken for 1 hr at r.t., after which a ninhydrin test is performed toestablish complete coupling. Final Fmoc deprotection is accomplished bytreatment of the resin with 20% v/v piperidine in DMF (5 mL) for 20 minsat r.t. The resin is drained, washed with DMF and CH₂Cl₂ and air dried.A cleavage cocktail of TFA:H₂O:DODT:iPr₃SiH (94:2.5:2.5:1% v/v, 10.0 mL)is added to the dry resin and the mixture shaken for 2 hr at r.t. Thecleavage cocktail is then treated with cold diethyl ether to precipitatethe crude peptide, which is centrifuged at 4000 rpm for 5 minutes. Thesupernatant is discarded and the pellet washed with diethyl ether,before repeating the spinning step. The ether phase is then discardedand the peptide dried with N₂ flow. The crude peptide is thenlyophilised from H₂O+0.1% TFA. The crude product is carried through tothe thiol-ene reaction step outlined below.

To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g loading, 0.2mmol scale) pre-swelled in 1:1 CH₂Cl₂:DMF is added a coupling mixture ofFmoc-Rink-Amide-OH (216 mg, 0.4 mmol), HBTU (151.8 mg, 0.4 mmol) andiPr₂NEt (140 μL, 0.8 mmol) in DMF (2 mL). The resin is shaken for 1 hrat r.t., after which a ninhydrin test indicated complete coupling. Thelinker amino group is then deprotected by treatment of the resin with20% v/v piperidine in DMF (5 mL) for 20 mins at r.t. The resin istransferred to a Tribute automated peptide synthesiser. Chain elongationup to and including the Ser(Trt) residue is performed using the generalautomated coupling method. Coupling of the Cys residue is performedmanually with addition of a mixture of Fmoc-Cys(Trt)-OH (235 mg, 0.4mmol), BOP (360 mg, 0.8 mmol), HOBt.H₂O (120 mg, 0.8 mmol) and2,4,6-collidine (120 μL, 0.8 mmol) in 1:1 CH₂Cl₂:DMF (2 mL). The resinis shaken for 1 hr at r.t., after which a ninhydrin test is performed toestablish complete coupling. After Fmoc deprotection, N-acetylation isperformed by adding acetic anhydride (50 μL) and iPr₂NEt (50 μL) in DMF(3 mL) to the resin. The resin is then shaken for 30 min at r.t., afterwhich a ninhydrin test is performed to establish no remaining freeamines. The resin is drained, washed with DMF and CH₂Cl₂ and air dried.A cleavage cocktail of TFA:H₂O:DODT:iPr₃SiH (94:2.5:2.5:1% v/v, 10.0 mL)is added to the dry resin and the mixture shaken for 2 hr at r.t. Thecleavage cocktail is then treated with cold diethyl ether to precipitatethe crude peptide, which is centrifuged at 4000 rpm for 5 minutes. Thesupernatant is discarded and the pellet washed with diethyl ether,before repeating the spinning step. The ether phase is then discardedand the peptide dried with N₂ flow. The crude peptide is thenlyophilised from H₂O+0.1% TFA. The crude product is carried through tothe thiol-ene reaction step outlined below.

Peptide Conjugates

To crude peptide 24 (25 mg, 32.6 μmol) and DMPA (3.3 mg, 13.1 μmol) in asolution of NMP (4 mL) is added vinyl palmitate (52.9 μL, 0.16 mmol).The resultant mixture is irradiated, with agitation, at 365 nm for 1 hrin a standard UV photochemical apparatus. The desired product 22 isdetected by mass analysis. The crude product 22 is purified viasemi-preparative RP HPLC on a Phenomenex Gemini C18 column running agradient of 5-65% MeCN:H₂O+0.1% TFA (3% MeCN per min, 50° C.). Massspectrometry is used to confirm the structure of the desired product 22.

The thiol-ene reaction of crude Cys-Ser-Lys-Lys-Lys-Lys-NH₂ with 5 eq.vinyl palmitate, 0.4 eq. DMPA in NMP, 1 hr irradiation at 365 nm givesthe desired product 20 (Pam-CSK₄), the identity of which is determinedby MS analysis.

To crude peptide 25 (20 mg) and DMPA (1.2 mg, 4.74 μmol) in a solutionof NMP (3 mL) is added vinyl palmitate (19.2 μL, 59.3 μmol). Theresultant mixture is irradiated, with agitation, at 365 nm for 1 hr in astandard photochemical apparatus. The desired product 26 is detected bymass analysis. The crude product 26 is purified via semi-preparative RPHPLC on a Phenomenex Gemini C18 column running a gradient of 5-65%MeCN:H₂O+0.1% TFA (3% MeCN per min, 50° C.). Mass spectrometry is usedto confirm the structure of the desired product 26 and the oxidisedMet(O) by-product.

1.5 General Method for Thiol-Ene Reaction on Peptides

To crude or purified peptide (10 mM), DTT (30 mM) and DMPA (4 mM) in asolution of DMSO is added vinyl palmitate (50 mM). The resultant mixtureis irradiated, with agitation, at 365 nm for 15 min in a standard UVphotochemical apparatus. The desired product is detected by ESI massanalysis. To achieve full conversion, further addition of DMPAphotoinitiator is sometimes required. The crude product is purified viasemi-preparative RP HPLC on a Phenomenex Gemini C18 column running agradient of 1-65% MeCN:H₂O+0.1% TFA (3% MeCN per min). Pooled fractionsare lyophilised to afford the pure products as white powders.

1.6 Discussion

The thermal reaction of Fmoc-Cys-OH with vinyl palmitate is conducted in1,2-dichloroethane, using 5 equivalents of alkene and 0.2 eq. of AIBN asradical initiator.

The reaction is performed under reflux (90 degrees) for 24 hrs.Microwave heating (100 W, 1 hr) is also used in certain embodiments. Thedesired product is detected by TLC. A number of by-products are in someembodiments also formed.

Photo-initiation of the reaction is conducted with 1 eq. vinyl palmitateand 0.2 eq. DMPA as the photo-initiator. Reactions are conducted in adegassed DMF:DCM solvent mixture, irradiated for 1 hr with 365 nm UVlight in a standard photo-chemical apparatus. Near complete conversionof the Fmoc-Cys-OH is monitored by TLC. Desirably, minimal by-productsare formed. Purification provides the product 200 in about reasonable tohigh yield. Using 2 eq. vinyl palmitate provides 200 in high yield afterpurification.

The thiol-ene reaction is carried out using NAc-CSK₄. The requiredpeptide motif 24 is synthesised as described above. Following attachmentof Rink-Amide linker to aminomethyl resin, the SK₄ sequence is built upusing automated Fmoc-SPPS (standard coupling conditions).Fmoc-Cys(Trt)-OH is then coupled manually using conditions to reduceepimerisation. N-acetylation is then carried out.

Mass analysis is used to establish whether by-product formation isoccurring upon cleavage of the peptide from the resin, due totert-butylation (+56) of cysteine. Repeating the synthesis of NAc-CSK₄utilising Fmoc-Ser(Trt)-OH, instead of Fmoc-Ser(t-Bu)-OH, is used incertain embodiments to produce a product free of the cysteine-alkylationproduct. The peptide is cleaved and then lyophilised.

The thiol-ene reaction of crude peptide 24 with vinyl palmitate is thencarried out. N-methylpyrrolidone (NMP) effectively solvates both thehydrophilic CSK₄ peptide and the hydrophobic vinyl palmitate molecule.

Thermal initiation using AIBN and microwave heating is carried out onboth crude and purified peptide using excess of vinyl palmitate (up to20 eq.). Photo-initiation of the reaction will in certain embodimentsprovide better results. Using crude peptide with DMPA asphoto-initiator, the reaction will in certain embodiments proceed tocompletion following 1 hr of irradiation (5 eq. vinyl palmitate, 0.4 eq.DMPA in 2 mL NMP). The desired product is confirmed by MS (ideally, >90%conversion, 60% purity by HPLC).

Advantageously, no purification after cleavage is required before thethiol-ene coupling. Purification by RP-HPLC is typically inefficient,with >50% loss of material being common. Generally, it is advantageousto reduce the number of HPLC purification steps required whereverpossible.

Purification of the N-acetylated monoacyl lipopeptide 22 is achieved bysemi-preparative RP-HPLC using a Phenomenex C18 column, running agradient of 5-95% MeCN:H₂O+0.1% TFA, 3% MeCN per min. The purifiedpeptide is then lyophilised to afford the desired product as a whitepowder.

Increasing the peptide concentration to 25 mM will in certainembodiments lead to a small decrease in by-product formation (forexample, >90% conversion, 80% purity by HPLC). Decreasing theconcentration to 5 mM will in certain cases have the opposite effect.

Carrying out the reaction in a mixture of NMP:H₂O:DMSO (4:2:1) in thepresence of glutathione (GSH)(3 eq.) with a peptide concentration of 5mM will in certain cases result in mixed disulfide formation (forexample, 50% conversion, 75% purity by HPLC). Using2,2′-(ethylenedioxy)diethanethiol (DODT)(3 eq.) in NMP with a peptideconcentration of 5 mM will in certain cases lead to a complex mixture ofproducts (for example, 80% conversion by HPLC).

In certain embodiments, addition of 3 eq. DTT to the reaction mixture(10 mM peptide in NMP) leads to no by-products resulting from vinylpalmitate telomerisation, or mixed disulfides, being observed and thereaction proceeds with high conversion (for example, >90% conversion,85% purity by HPLC). Using DTT it is also possible to conduct thereaction (25 mM peptide) in DMSO, a benign and more versatile solvent(for example, to achieve 90% conversion, >95% purity by HPLC).

The thiol-ene reaction is also carried out using non-acetylated analogueCSK₄. Synthesis of the CSK₄ motif is carried out utilising the proceduredescribed above. The peptide is then cleaved from resin and lyophilised.In certain embodiments, the thiol-ene reaction of the crude product withvinyl palmitate proceeds smoothly using 5 eq. vinyl palmitate, 0.4 eq.DMPA in NMP, 1 hr irradiation at 365 nm to give the desired product 20(Pam-CSK₄), the identity of which is confirmed by MS analysis.

The thiol-ene reaction of vinyl palmitate with a long peptide comprisingEBV LMP2 epitopes, LMP2 S-1 [SEQ ID NO: 5], is also carried out.

The LMP2 S-1 sequence is built up by automated Fmoc-SPPS, using standardconditions. A K₄ tag and a serine residue are then coupled to theN-terminus of the sequence, depicted herein as SEQ ID NO: 4. Thepeptidyl resin is then removed from the synthesiser and the cysteineresidue coupled manually, using standard conditions. N-acetylation isthen carried out. The peptide is then cleaved from the resin andlyophilised to give a white powder in good yield.

The thiol-ene reaction of the unprotected peptide 25 and vinyl palmitateis carried out using photo-initiation, as described for peptides 20 and22. Mass analysis is performed to establish conversion to thepalmitoylated product 26. Purification is accomplished bysemi-preparative RP-HPLC using a Phenomenex C18 column, running agradient of 5-95% MeCN:H₂O with 0.1% TFA. The purified peptide islyophilised to provide the desired product as a white powder, along withthe corresponding Met(O) product.

The thiol-ene reaction of crude 25 with vinyl palmitate is also carriedout following the general procedure described above. ESI-MS and HPLCanalysis are performed to establish good conversion to the palmitoylatedproduct 26. Purification is accomplished by semi-preparative RP-HPLC, togive the desired product, for example in >95% purity.

Example 2. Biological Activity of Peptide Conjugates 20, 22, and 26

2.1 Procedures

Activation of Human Monocytes in Whole Blood

100 μl of heparinised whole blood (WB) is incubated with 100 nM, 1 μMand 10 μM of each compound, in duplicate, and incubated overnight at 37°C. in a 5% CO₂ humidified incubator. Pam₃CSK₄ (10 μM; EMCMicrocollections) is used as a positive control. To detect activation ofmonocytes, WB samples are stained with anti-CD14-FITC,anti-HLA-DR-Alexa700, anti-CD80-PE-Cy7, anti-CD40-PE, anti-CD86-APC,anti-CD16-APC-Cy7 (all from Biolegend) for 20 mins at RT, protected fromlight. Following incubation, 2 ml of BD FACS lyse (BD Biosciences) isadded, incubated for 15 mins at RT, then washed twice with ice cold washbuffer (PBS, 1% Human Serum). Data acquisition is performed on a BD FACSAria II (Becton Dickinson) and analysed using FlowJo software version7.6.5 (TreeStar). CD80 receptor expression on monocytes is detected bygating on CD14+ HLADR+ cells.

Toll-Like Receptor 2 (TLR2) Agonism Using HekBlue Cells

HEK-Blue™-hTLR2 and HEK-Blue™-mTLR2 are purchased from Invivogen. TheseHEK-Blue cells are produced by co-transfection of both reporter geneSEAP (secreted embryonic alkaline phosphatase) and either human ormurine TLR2, respectively. The SEAP reporter gene is under the controlof the IFN-β minimal promoter fused to five AP-1 and five NFkB bindingsites. Cells are cultured according to manufacturer's instructions. Onthe day of the assay, the constructs are added at the selectedconcentrations in 20 μl volume of endotoxin free water in a 96-wellplate. HEK-Blue™-hTLR2 or HEK-Blue™-hTLR2 cells are resuspended at˜2.83×10⁴ cells/ml in HEK-Blue™ Detection medium and immediately add 180ml of the cell suspension (˜5×10⁴ cells per well.) The cells areincubated overnight at 37° C. in 5% CO₂. SEAP expression was quantifiedusing an EnSpire plate reader (PerkinElmer) at 635 nM.

Method for Detection of IL-8 Secretion from TLR2-Transiently TransfectedHek293 Cells

Hek-293 cells are plated 3×10⁴ cells in 50 μl per well in 96-well platewith DMEM containing 10% FBS (the medium is not supplemented withantibiotics). Cells are transfected with either a combination ofpFLAG-TLR2 plasmid and pcDNA3.1 (a kind gift from Shimizu, as reportedin Shimizu, T., Y. Kida and K. Kuwano (2005). “A dipalmitoylatedlipoprotein from Mycoplasma pneumoniae activates NF-kappa B throughTLR1, TLR2, and TLR6.” J Immunol 175(7): 4641-4646), or the controlplasmid only (pcDNA3.1). Master mix of Lipofectamine/DNA complexes areconstituted in Opti-MEM at 100 ngDNA in 0.3 μl Lipofectamine in a volumeof 50 μl per sample. Following an incubation of 20 mins, the plasmid mixwas added to the cells. Protein expression was induced for 24 hoursprior to the addition of constructs.

The constructs are added to the wells at the selected concentrations tomake a final volume of 200 μl per well. Following 18-hours ofstimulation, the supernatant was harvested from each sample and storedat −20° C. until required. IL-8 secretion is determined by CytometricBead Array (BD Biosciences) according to manufacturer's protocol,optionally with one modification: 25 μl of conditioned medium can beused instead of 50 μl. To accurately determine the concentration ofsecreted IL-8, an 11-point standard curve (1-5000 ng/ml) is performed.Samples are analysed using a BD-FACS Aria II (BD Biosciences) and thedata is then analysed using FCAP ARRAY Software (version 1.0.1).

2.2 Discussion

The bioactivity of lipopeptides 20, 22, and 26, is assessed by flowcytometry to measure up-regulation of the co-stimulatory molecule CD80on human monocytes in fresh blood samples.

Monocytes are identified in donor samples by characteristic cell surfacemarkers, and the expression of CD80 determined before and after exposureto each compound at three dosages, with commercially available Pam3CSK4(10 μM) serving as a positive control.

Results showing test lipopeptides 20, 22 and 26 strongly upregulateexpression of CD80 at all doses tested, for example at equivalent orgreater potency to positive control Pam3CSK4, are indicative ofeffective TLR agonism.

Results showing test lipopeptides 20, 22 and 26 demonstrate TLR agonismin HekBlue™ and IL-8 reporter systems, for example, titratable TLRagonism, are indicative of effective TLR agonism.

Results showing high potency of 20, 22 and 26 are supportive of thefinding that conjugation of antigenic peptides does not affect TLR2agonism.

Example 3. Preparation of Conjugates 200, 120, 121, 110-112, 112A, and113-116

3.1 General Details

Protected amino acids and coupling reagents are purchased fromGL-Biochem (Shanghai). The resins used in the solid-supported synthesesare preloaded tentagel resins from Rapp Polymere GmbH (Tuebingen) andother solvents and reagents are obtained from Sigma (St Louis, Mo.) andNovabiochem.

The peptide syntheses described below are carried out using standarditerative Fmoc Solid-Phase Peptide Synthesis techniques on a Tributepeptide synthesiser (Protein Technologies International, Tucson, Ariz.).A typical deprotection and coupling cycle carried out on a 0.1 mmolscale entails removal of the Fmoc protecting group from the resin-boundamino-acid using two treatments of 20% piperidine in DMF (4 mL×5 min)then washing the resin with DMF. In a separate vessel the Fmoc aminoacid (0.5 mmol) and coupling agent(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU), 0.45 mmol) are dissolved in DMF (1.5mL) and base (4-methylmorpholine, 1 mmol) is added. After mixing for 1minute, this solution is transferred to the resin, which is agitated atRT for 1 hour, drained and washed.

Cleavage of the peptide (0.1 mmol scale) is achieved by suspending theresin in 5 mL trifluoroacetic acid (TFA) containing 5% (v/v)ethanedithiol (EDT) and agitating at room room temperature for 3 hours.Triisopropylsilane (TIPS) is then added to 1% (v/v) and agitationcontinued for a further five minutes before draining the TFA intochilled diethyl ether (40 mL). The precipitated material is pelleted bycentrifugation, the ether discarded, the pellet washed once with ether(25 mL) and air-dried or lyophilised.

Reverse phase (RP)-HPLC is carried out using a Dionex Ultimate 3000 HPLCsystem. For semi-preparative purifications, a peptide sample is injectedinto a reverse-phase Phenomenex Gemini C18 column (5μ, 110 Å; 10×250 mm)equilibrated in a suitable mixture of eluent A (water/0.1% TFA) andeluent B (MeCN/0.1% TFA) then an increasing gradient of eluent B isgenerated to elute the constituent components. Analytical HPLC isperformed similarly, using a Phenomenex Gemini C18 column (3μ, 110 Å;4.6×150 mm).

Low-resolution mass spectra are obtained using an Agilent Technologies6120 Quadrapole mass spectrometer.

NMR spectra are obtained using a Bruker BRX400 spectrometer operating at400 MHz for ¹H NMR and at 100 MHz for ¹³C NMR.

In the amino acid conjugates and peptide conjugates described below theabbreviations AcN-C(Pam-1)- and H₂N—C(Pam-1)- means

wherein R is Ac or H as appropriate.3.2 Preparation of Peptide Conjugates by Direct ConjugationPeptides

Peptides 100 and 102 (comprising LMP2 S-2, SEQ ID NO: 10), 103 and 104(comprising LMP2 S-3, SEQ ID NO: 15), and 105 and 106 (comprising LMP25-2, SEQ ID NO: 30), as depicted in Table 3 below are synthesised asdescribed and depicted below (Scheme 1).

Following synthesis of the peptide sequence up to the penultimate aminoacid using iterative Fmoc-SPPS, Fmoc-cysteine is introduced as theN-terminal residue of the on-resin peptide by reaction withFmoc-Cys(Trt)-OH, HATU, and 4-methylmorpholine in DMF. The Fmoc group isremoved using 20% piperidine in DMF. As required, the resulting aminegroup is converted to an acetamide by treatment with a mixture of 20%acetic anhydride in DMF (2 mL) and 4-methylmorpholine (1 mmol).

Following cleavage of the peptide from resin with TFA/EDT and itsprecipitation in ether, the solid is dissolved in 1:1 water/MeCN andlyophilised. If the peptide contained a methionine residue the solutionis heated at 60° C. for 1 hour prior to freeze-drying to reverse anyS-alkylation that may have occurred during cleavage. The peptides arethen purified by RP-HPLC to give material of >95%.

TABLE 3 SEQ ID Sequence NO: 100 AcHN-CSKKKSLYLGLQHDGNDGLPPPPYSPRDDSSQHI104 YEEA(Ac)-C(O)NH2 102 AcHN-CSKKKKSLYLGLQHDGNDGLPPPPYSPRDDSSQH 104IYEEA-OH 103 H₂N-CSKKKKSDYQPLGTQDQSLYLGLQHDGNDGL-OH 105 104AcHN-CSKKKKSDYQPLGTQDQSLYLGLQHDGNDGL-OH 105 105H₂N-CSKKKKLMLLWTLVVLLICSSCSSCPLSKILLARL 106 FLYALALLLLA-OH 106AcHN-CSKKKK LMLLWTLVVLLICSSCSSCPLSKILLA 106 RLFLYALALLLLA-OHPeptide Conjugates

The thiol-ene reaction is then performed on peptides 100 and 102-106 togenerate the corresponding peptide conjugates 110, 112, and 113-116(Table 4).

DMPA (2.6 mg), dithiothreitol (9.2 mg), and vinyl palmitate (40 mg,mmol) are dissolved in degassed NMP (2 mL). 100 μL of this solution isthen added to 1 μmol of the peptide weighed into a small polypropylenevessel to give a solution containing 10 mM peptide, 5 mM DMPA, 30 mM DTTand 50 mM vinyl palmitate. NMP is compatible with the reactionconditions and effectively solvates all of the components of thereaction mixture.

The reaction vessel is flushed with nitrogen and the vigorously stirredmixture irridiated with a hand-held 6 watt UV lamp (Spectronics, NY)operating at 365 nm. After 30 minutes the reaction is analysed by HPLCand conversion to the desired product is determined. The product is thenisolated by RP-HPLC and unreacted starting material recovered.

The peptides with non-acetylated N-terminal cysteine will in certainembodiments form significant amounts of the disulfide dimer, despite thepresence of the reducing agent DTT. This is not typically observed withthe corresponding N-acetylated peptides.

Peptide conjugates 110 and 113-116 are also prepared from peptides 100and 103-106 by the following alternative procedure (Table 4B).

In this procedure, tert-butyl mercaptan (tBuSH) thiol is used in placeof DTT. In certain embodiments, this result in increased and cleanerconversion of the substrate peptides to the desired peptide conjugate.

Trifluoroacetic acid (TFA) is also introduced to the reaction mixture.In certain embodiments, this further improves the reaction profile. Theformation of oligomers, minor by-products formed by reaction of theproduct peptide conjugate with a second molecule of vinyl palmitate togive a bis-palmitoylated species, is largely suppressed by the additionof TFA.

Any apparent propensity of methionine to oxidise to the correspondingsulfoxide under these conditions, can be resolved by lypophilising thecrude product mixtures of those peptides possessing methionine groups,followed by dissolution in TFA and treatment with tetrabutylammoniumiodide to reduce methionine oxide back to methionine.

A typical procedure is as follows. DMPA (6.5 mg) is dissolved indegassed NMP (0.5 mL) and tert-butyl mercaptan (17 μL) added and in aseparate vessel vinyl palmitate (11.3 mg) is dissolved in degassedN-methylpyrrolidinone (NMP) (0.5 mL). The peptide (1 μmol) is weighedinto a small polypropylene vessel equipped with a small stirrer and 10μL of the DMPA/tBuSH solution added followed by 100 μL of the vinylpalmitate solution, to give a solution of approximately 10 mM peptide, 5mM DMPA, 30 mM DTT and 80 mM vinyl palmitate. TFA (5.5 μL) is thenadded, to give a 5% solution. The reaction vessel is flushed withnitrogen and the vigorously stirred mixture irradiated with a hand-held6 watt UV lamp (Spectronics, NY) operating at 365 nm. After 20 minutesfurther DMPA (10 μL) and vinyl palmitate (50 μL) are added andirradiation continued for 20 min.

For those peptides containing methionine, water (0.5 mL) and MeCN (0.5mL) are added and the mixture lyophilised. The resultant solid isdissolved in neat TFA (150 μL), cooled to 0° C. andtetra-n-butylammonium iodide (3.7 mg, 10 μmol) in 25 μL TFA is added.After 1 minute chilled diethyl ether (0.5 mL) is added to precipitatethe reduced lipopeptide, which is pelleted by centrifugation andlyophilised.

The reactions are analysed by HPLC to show conversion to the desiredproducts (Table 4B), which are then isolated by RP-HPLC.

TABLE 4 SEQ ID Sequence NO: 110 AcHN-C(Pam-1)SKKKSLYLGLQHDGNDGLPPPPYSPRD104 DSSQHIYEEA(Ac)-C(O)NH₂ 112 AcHN-C(Pam-1)SKKKKSLYLGLQHDGNDGLPPPPYSPR104 DDSSQHIYEEA-OH^(a) 113 H₂N-C(Pam-1)SKKKKSDYQPLGTQDQSLYLGLQHDGND 105GL-OH 114 AcHN-C(Pam-1)SKKKKSDYQPLGTQDQSLYLGLQHDGN 105 DGL-OH 115H₂N-C(Pam-1)SKKKKLMLLWTLVVLLICSSCSSCPLSK 106 ILLARLFLYALALLLLA-OH 116AcHN-C(Pam-1)SKKKKLMLLWTLVVLLICSSCSSCPLS 106 KILLARLFLYALALLLLA-OH

TABLE 4B SEQ ID Sequence NO: 110AcHN-C(Pam-1)SKKKSLYLGLQHDGNDGLPPPPYSPRD 104 DSSQHIYEEA(Ac)-C(O)NH2 113H2N-C(Pam-1)SKKKKSDYQPLGTQDQSLYLGLQHDGND 105 GL-OH 114AcHN-C(Pam-1)SKKKKSDYQPLGTQDQSLYLGLQHDGN 105 DGL-OH 116AcHN-C(Pam-1)SKKKKLMLLWTLVVLLICSSCSSCPLS 106 KILLARLFLYALALLLLA-OH3.3 Preparation of Amino Add Conjugates

Amino acid conjugates 200, 120, and 121 are prepared from N-α-Fmoc-,N-α-acetyl-, and N-α-Boc-protected cysteine, respectively, as describedand depicted below (Scheme 2).

Solid N-α-protected cysteine is dissolved or suspended to aconcentration of 100 mg/mL in the indicated solvent (Table 5) and vinylpalmitate (1.5 molar equivalents) added followed by the indicatedquantity of initiator. For reactions conducted under photolyticconditions the solution is prepared in a polypropylene vessel, DMPAadded in the indicated molar proportions (Table 5) and the stirredmixture then irradiated at 365 nm. For reactions carried out underthermal conditions, the solution is prepared in a glass tube, theindicated quantity of AIBN (azobisisobutyronitrile) added and thestirred mixture heated either in an oil bath or in a microwave oven.

Reaction progress is monitored using thin-layer chromatography and isallowed to proceed to completion based on consumption of the cysteinestarting material. The solvent is then removed and the residue purifiedby flash column chromatography on silica gel, eluting with hexane/ethylacetate mixtures. The identities of Fmoc-Cys(Pam-1)-OH (200),Ac-Cys(Pam-1)-OH (120) and Boc-Cys(Pam-1)-OH (121), are confirmed by ¹Hand ¹³C NMR and by mass spectrometry.

The conjugation reaction is carried out under a variety of conditions,which are summarized in Table 5.

TABLE 5 N-α-protecting Initiator Time Entry group (mol eq.) SolventConditions^(a) (min) 1 Fmoc DMPA (0.2) DCM hu (365 nm) 60 2 Fmoc DMPA(1) DCM hu (365 nm) 60 3 Fmoc AIBN (1) DCM Microwave 80 (70° C.) 4 AcDMPA (0.2) DCM hu (365 nm) 60 5 Ac DMPA (0.2) DCM hu (365 nm), 60 DTT 6Ac DMPA (1) DCM hu (365 nm) 60 7 Ac AIBN (1) DCM Microwave 80 (70° C.) 8Boc DMPA (0.2) DCM hu (365 nm) 60 9 Boc DMPA (1) DCM hu (365 nm) 60 10Boc AIBN (1) DCM Microwave 80 (70° C.) ^(a)UV irradiation used ahand-held Spectronics 6 watt lamp operating at 365 nm; microwavereactions are carried out using a CEM Discover microwave reactoroperating at 100 w and 70° C.

The use of photolytic conditions or thermal conditions will in certainembodiments generate the desired products with desirably high yields.

3.4 Preparation of Peptide Conjugates Via Coupling of Amino AddConjugates

Peptide conjugates 110-116 are prepared as described and depicted below(Scheme 3).

The desired peptide sequence is synthesised using standard iterativeFmoc SPPS techniques using a Tribute peptide synthesiser as previouslydescribed. After coupling the penultimate amino acid residue, theresin-bound peptide chain is then derivatised with the amino acidconjugate N-Fmoc-Cys(Pam-1)-OH 200 using PyBOP and collidine in DMF. TheFmoc group is then removed using 20% piperidine in DMF.

The resulting peptide is then cleaved from resin using TFA/EDT, withconcomitant removal of protecting groups, to afford peptide conjugates111, 113 and 115.

Alternatively, the resulting peptide is converted to the correspondingacetamide by treatment with a mixture of 20% acetic anhydride in DMF (2mL) and 4-methylmorpholine (1 mmol) and then cleaved from resin toafford peptide conjugates 110, 112, 114 and 116.

Alternatively, the resin-bound peptides are derivatised with either theamino acid conjugate N-Boc-Cys(Pam-1)-OH 121 or N—Ac-Cys(Pam-1)-OH 120.On cleavage from resin this affords the peptide conjugates 110-116directly, without the additional manipulations necessary due to the Fmocgroup.

The conditions for coupling of the amino acid conjugate advantageouslyreduces the propensity of the α-carbon of the amino acid to racemise onactivation. The amino acid conjugate (0.075 mmol) and PyBOP(benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate)(0.1 mmol) are combined and dissolved in DMF (0.3 mL). Neat2,4,6-trimethylpyridine (0.1 mmol) is added and after mixing for 30seconds the solution transferred to 0.025 mmol of resin, which is thenagitated for 90 minutes, drained and washed (DMF).

The peptide is then cleaved by agitating 0.015 mmol of the resin in 1 mLof trifluoroacetic acid containing 5% (v/v) ethanedithiol at roomtemperature for 3 hours. The supernatant is then drained through asinter into chilled diethyl ether (10 mL) and the resin is washed with afurther 1 mL of TFA, which is also added to the ether.

The precipitated material is pelleted by centrifugation and the pelletwashed once with ether (5 mL) before being dissolved in 1:1 MeCN/Water(+0.1% tfa) and lyophilised. If the peptide contained a methionineresidue the solution is heated at 60° C. for 1 hour prior tofreeze-drying. The peptides are then purified (>95%) by RP-HPLC andtheir identities confirmed by analytical RP-HPLC and mass spectrometry.

Example 4. Analysis of Peptides LMP2 S1 & LMP2 S2

1.1 General Details

Peptides LMP2 S1 (DRHSDYQPLGTQDQSLYLGLQHDGNDGL, SEQ ID NO: 5) and LMP2S2 (SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA, SEQ ID NO: 10) were synthesisedessentially as described above. The peptides were then purified (>95%)by RP-HPLC and their identities confirmed by analytical RP-HPLC and massspectrometry.

Results

FIG. 1 shows an RP-HPLC trace of LMP2 S1, under the followingconditions: column: Phenomenex Gemini C18 (5μ 110 Å, 4.6×150 mm);gradient: 0-1 min, 5% B then 5% B to 65% B over 30 min., eluting at 1mL/min., Rt 14.4 min.

ESI-MS trace m/z [M+2H]2+=1572.7.

FIG. 2 shows an RP-HPLC trace of LMP2 S2, under the followingconditions: column: Phenomenex Gemini C18 (5μ 110 Å, 4.6×150 mm);gradient: 0-1 min, 1% B then 1% B to 61% B over 30 min., eluting at 1mL/min., Rt 16.0 min.

ESI-MS trace m/z [M+2H]2+=1448.2.

Example 5. Analysis of Conjugated LMP2 S4(SK4)

1.1 General Details

Peptide LMP2 S4(SK4)

(SKKKKSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA, SEQ ID NO: 19) wassynthesised, acetylated and conjugated to Pam1Cys essentially asdescribed above, to give Pam1-C(Ac)SK4-LMP2 S4 [SEQ ID NO: 107]:

The identity of the crude peptide was then confirmed by analyticalRP-HPLC and mass spectrometry.

Results

FIG. 3 shows an RP-HPLC trace of Pam1-C(Ac)SK4-LMP2 S4, under thefollowing conditions: column: Phenomenex Gemini C18 (5μ 110 Å, 4.6×150mm); gradient: 0-1 min, 5% B then 5% B to 65% B over 30 min., eluting at1 mL/min., Rt 21.6 min.

ESI-MS trace m/z [M+3H]3+=1977.0 It is not the intention to limit thescope of the invention to the abovementioned examples only. As would beappreciated by a skilled person in the art, many variations are possiblewithout departing from the scope of the invention.

INDUSTRIAL APPLICABILITY

The peptides, and amino acid and peptide conjugates and compositions andconstructs of the invention find application in the pharmaceutical andmedical fields, including application in methods of eliciting immuneresponses in a subject and methods of vaccinating a subject. Forexample, medicaments comprising the peptides or amino acid and peptideconjugates directed to treating Epstein Barr Virus (EBV) associateddiseases, such as Hodgkin's Disease (HD) or Nasopharangeal Carcinoma(NPC), including, for example, self-adjuvating vaccines comprising oneor more epitopes from EBV Latent Membrane Protein 2 (LMP2), areparticularly contemplated.

The invention claimed is:
 1. A compound of the formula (V):

wherein m is an integer from 0 to 4; n is 1 or 2; R1 and R2 at eachinstance of m are each independently hydrogen, C1-6alkyl, orC3-6cycloalkyl; R3, R4, R5, R8, and R9 are each independently hydrogen,C1-6alkyl, or C3-6cycloalkyl; or R9 is an amino protecting group,L3-C(O), or A2; R6 and R7 at each instance of n are each independentlyhydrogen, C1-6alkyl, or C3-6cycloalkyl, L1 is C5-21alkyl orC4-20heteroalkyl; L3 is C1-6alkyl or C3-6cycloalkyl; A1 and A2 are eachindependently an amino acid or a peptide; or A1 is OH or OP1, wherein P1is a carboxyl protecting group; and wherein A1 comprises one or more EBVLMP2 epitopes or R9 is A2 and comprises one or more EBV LMP2 epitopes,or wherein (i) A1 comprises one or more peptides selected from the groupconsisting of SEQ ID NOs: 1-101, (ii) R9 is A2 and comprises one or morepeptides selected from the group consisting of SEQ ID NOs: 1-101, or(iii) A1 comprises one or more peptides selected from the groupconsisting of SEQ ID NOs: 1-101 and R9 is A2 and comprises one or morepeptides selected from the group consisting of SEQ ID NOs: 1-101;wherein any alkyl, cycloalkyl or heteroalkyl present in any of R1, R2,R4, R5, R6, R7, R8, R9, L1, and L3 and any cycloalkyl in R3 isoptionally substituted with one or more substituents independentlyselected from the group consisting of halo, CN, NO₂, OH, NH₂, NH(C1-6alkyl), N (C1-6alkyl) (C1-6alkyl), C1-6haloalkyl, C1-6haloalkoxy,C(O)NH₂, C(O)NH(C1-6alkyl), C(O)N(C1-6alkyl) (C1-6alkyl),SO2(C1-6alkyl), O(C1-6alkyl), S(C1-6alkyl), S(O)(C1-6alkyl),C(O)(C1-6alkyl), and C1-6aliphatic, or a pharmaceutically acceptablesalt or solvate thereof.
 2. The compound of claim 1, wherein R9 isindependently hydrogen, C1-6alkyl, or C3-C6cycloalkyl; or R9 is L3-C(O)or A2; and A1 and A2 are each independently a peptide; or A1 is OH;provided that: at least one of A1 and A2 comprises an EBV LMP2 epitope;and when R9 is not A2, A1 is a peptide.
 3. The compound of claim 1,wherein L1 is C5-21alkyl or linear C15alkyl.
 4. The compound of claim 1,wherein m is an integer from 0 to 2 or m is 0 or
 1. 5. The compound ofclaim 1, wherein m is
 0. 6. The compound of claim 1, wherein R1 and R2at each instance of m are each independently hydrogen.
 7. The compoundof claim 1, wherein R3 is hydrogen.
 8. The compound of claim 1, whereinR4 and R5 are each hydrogen.
 9. The compound of claim 1, wherein R6 andR7 Eire each hydrogen.
 10. The compound of claim 1, wherein R8 ishydrogen and R9 is hydrogen, an amino protecting group, L3-C(O), or A2.11. The compound of claim 1, wherein L3 is Me.
 12. The compound of claim1, wherein the peptide comprises an epitope selected from the groupconsisting of the amino acid sequence of any one of SEQ ID NOs: 76-101.13. The compound of claim 1, wherein A1 is serine or a peptidecomprising serine as the first N-terminal amino acid residue.
 14. Thecompound of claim 1, wherein A1 and/or A2 is a peptide comprising asolubilising group comprising an amino acid sequence comprising two ormore hydrophilic amino acid residues in the peptide chain.
 15. Thecompound of claim 1 wherein the peptide comprises, consists essentiallyof, or consists of an amino acid sequence selected from the groupconsisting of a. 8 or more contiguous amino acid residues from thesequence of any one of SEQ ID NO:1 to 101; b. 10 or more contiguousamino acid residues from the sequence of any one of SEQ ID NO:1 to 101;c. 12 or more contiguous amino acid residues from the sequence of anyone of SEQ ID NO:1 to 101; d. 15 or more contiguous amino acid residuesfrom the sequence of any one of SEQ ID NO:1 to 101; e. 20 or morecontiguous amino acid residues from the sequence of any one of SEQ IDNO:1 to 101; f. the sequence of any one of SEQ ID NOs: 1 to 101, g. 8 ormore contiguous amino acid residues from the sequence of any one of SEQID NO:1 to 93; h. 10 or more contiguous amino acid residues from thesequence of any one of SEQ ID NO:1 to 93; 12 or more contiguous aminoacid residues from the sequence of any one of SEQ ID NO:1 to 93; 15 ormore contiguous amino acid residues from the sequence of any one of SEQID NO:1 to 93; k. 20 or more contiguous amino acid residues from thesequence of any one of SEQ ID NO:1 to 93; l. the sequence of any one ofSEQ ID NOS: 1 to 93, m. 8 or more contiguous amino acid residues fromthe sequence of any one of SEQ ID NO:1 to 75; n. 10 or more contiguousamino acid residues from the sequence of any one of SEQ ID NO:1 to 75;o. 12 or more contiguous amino acid residues from the sequence of anyone of SEQ ID NO:1 to 75; p. 15 or more contiguous amino acid residuesfrom the sequence of any one of SEQ ID NO:1 to 75; q. 20 or morecontiguous amino acid residues from the sequence of any one of SEQ IDNO:1 to 75; r. the sequence of any one of SEQ ID NOs: 1 to 75, and s.any combination of two or more of (a) to (r) above.
 16. A pharmaceuticalcomposition comprising an effective amount of a peptide conjugate ofclaim 1 or a pharmaceutically acceptable salt or solvate thereof, or anycombination thereof, and a pharmaceutically acceptable carrier.
 17. Amethod of vaccinating or eliciting an immune response in a subjectcomprising administering to the subject an effective amount of a peptideconjugate of claim 1 or a pharmaceutically acceptable salt or solvatethereof.
 18. A method for making a peptide conjugate of claim 1, themethod comprising (A) reacting a lipid-containing conjugation partner,and an amino acid-comprising conjugation partner, under conditionseffective to conjugate the lipid-containing conjugation partner to theamino acid-comprising conjugation partner by the hydrothiolation of acarbon-carbon double bond with a thiol, the method further comprisingcoupling the amino acid of the amino acid conjugate to an amino acid ora peptide to provide a peptide conjugate, and wherein the peptideconjugate comprises one or more EBV LMP2 epitopes; or (B) reactinglipid-containing conjugation partner, and a peptide-comprisingconjugation partner, wherein the peptide-comprising partner comprisesone or more EBV LMP2 epitopes, under conditions effective to conjugatethe lipid-containing conjugation partner to the peptide-comprisingconjugation partner by the hydrothiolation of a carbon-carbon doublebond with a thiol; or (C) providing a lipididated amino acid or peptide,and coupling the lipidated amino acid or peptide to one or more aminoacids or peptides to provide a peptide conjugate, and wherein thepeptide conjugate comprises one or more EBV LMP2 epitopes.